Gene expression profile algorithm and test for determining prognosis of prostate cancer

ABSTRACT

The present invention provides algorithm-based molecular assays that involve measurement of expression levels of genes, or their co-expressed genes, from a biological sample obtained from a prostate cancer patient. The genes may be grouped into functional gene subsets for calculating a quantitative score useful to predict a likelihood of a clinical outcome for a prostate cancer patient.

RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 14/221,556, filed Mar. 21, 2014, which is a continuation of U.S. application Ser. No. 13/752,199, filed Jan. 28, 2013, now U.S. Pat. No. 8,725,426, issued May 13, 2014, which claims the benefit of priority of U.S. Provisional Application Nos. 61/593,106, filed Jan. 31, 2012; 61/672,679, filed Jul. 17, 2012; and 61/713,734, filed Oct. 15, 2012, all of which are hereby incorporated by reference in their entirety.

TECHNICAL FIELD

The present disclosure relates to molecular diagnostic assays that provide information concerning gene expression profiles to determine prognostic information for cancer patients. Specifically, the present disclosure provides an algorithm comprising genes, or co-expressed genes, the expression levels of which may be used to determine the likelihood that a prostate cancer patient will experience a positive or a negative clinical outcome.

INTRODUCTION

The introduction of prostate-specific antigen (PSA) screening in 1987 has led to the diagnosis and aggressive treatment of many cases of indolent prostate cancer that would never have become clinically significant or caused death. The reason for this is that the natural history of prostate cancer is unusual among malignancies in that the majority of cases are indolent and even if untreated would not progress during the course of a man's life to cause suffering or death. While approximately half of men develop invasive prostate cancer during their lifetimes (as detected by autopsy studies) (B. Halpert et al, Cancer 16: 737-742 (1963); B. Holund, Scand J Urol Nephrol 14: 29-35 (1980); S. Lundberg et al., Scand J Urol Nephrol 4: 93-97 (1970); M. Yin et al., J Urol 179: 892-895 (2008)), only 17% will be diagnosed with prostate cancer and only 3% will die as a result of prostate cancer. Cancer Facts and Figures. Atlanta, Ga.: American Cancer Society (2010); J E Damber et al., Lancet 371: 1710-1721 (2008).

However, currently, over 90% of men who are diagnosed with prostate cancer, even low-risk prostate cancer, are treated with either immediate radical prostatectomy or definitive radiation therapy. M R Cooperberg et al., J Clin Oncol 28: 1117-1123 (2010); M R Cooperberg et al., J Clin Oncol 23: 8146-8151 (2005). Surgery and radiation therapy reduce the risk of recurrence and death from prostate cancer (AV D'Amico et al., Jama 280: 969-974 (1998); M Han et al., Urol Clin North Am 28: 555-565 (2001); W U Shipley et al., Jama 281: 1598-1604 (1999); A J Stephenson et al., J Clin Oncol 27: 4300-4305 (2009)), however estimates of the number of men that must be treated to prevent one death from prostate cancer range from 12 to 100. A Bill-Axelson et al., J Natl Cancer Inst 100: 1144-1154 (2008); J Hugosson et al., Lancet Oncol 11: 725-732 (2010); L H Klotz et al., Can J Urol 13 Suppl 1: 48-55 (2006); S Loeb et al., J Clin Oncol 29: 464-467 (2011); F H Schroder et al., N Engl J Med 360: 1320-1328 (2009). This over-treatment of prostate cancer comes at a cost of money and toxicity. For example, the majority of men who undergo radical prostatectomy suffer incontinence and impotence as a result of the procedure (M S Litwin et al., Cancer 109: 2239-2247 (2007); M G Sanda et al., N Engl J Med 358: 1250-1261 (2008), and as many as 25% of men regret their choice of treatment for prostate cancer. FR Schroeck et al., Eur Urol 54: 785-793 (2008).

One of the reasons for the over-treatment of prostate cancer is the lack of adequate prognostic tools to distinguish men who need immediate definitive therapy from those who are appropriate candidates to defer immediate therapy and undergo active surveillance instead. For example, of men who appear to have low-risk disease based on the results of clinical staging, pre-treatment PSA, and biopsy Gleason score, and have been managed with active surveillance on protocols, 30-40% experience disease progression (diagnosed by rising PSA, an increased Gleason score on repeat biopsy, or clinical progression) over the first few years of follow-up, and some of them may have lost the opportunity for curative therapy. H B Carter et al., J Urol 178: 2359-2364 and discussion 2364-2355 (2007); M A Dall'Era et al., Cancer 112: 2664-2670 (2008); L Klotz et al., J Clin Oncol 28: 126-131 (2010). Also, of men who appear to be candidates for active surveillance, but who undergo immediate prostatectomy anyway, 30-40% are found at surgery to have higher risk disease than expected as defined by having high-grade (Gleason score of 3+4 or higher) or non-organ-confined disease (extracapsular extension (ECE) or seminal vesicle involvement (SVI)). S L et al., J Urol 181: 1628-1633 and discussion 1633-1624 (2009); C R Griffin et al., J Urol 178: 860-863 (2007); P W Mufarrij et al., J Urol 181: 607-608 (2009).

Estimates of recurrence risk and treatment decisions in prostate cancer are currently based primarily on PSA levels and/or clinical tumor stage. Although clinical tumor stage has been demonstrated to have a significant association with outcome, sufficient to be included in pathology reports, the College of American Pathologists Consensus Statement noted that variations in approach to the acquisition, interpretation, reporting, and analysis of this information exist. C. Compton, et al., Arch Pathol Lab Med 124:979-992 (2000). As a consequence, existing pathologic staging methods have been criticized as lacking reproducibility and therefore may provide imprecise estimates of individual patient risk.

SUMMARY

This application discloses molecular assays that involve measurement of expression level(s) of one or more genes or gene subsets from a biological sample obtained from a prostate cancer patient, and analysis of the measured expression levels to provide information concerning the likelihood of a clinical outcome. For example, the likelihood of a clinical outcome may be described in terms of a quantitative score based on clinical or biochemical recurrence-free interval, overall survival, prostate cancer-specific survival, upstaging/upgrading from biopsy to radical prostatectomy, or presence of high grade or non-organ confined disease at radical prostatectomy.

In addition, this application discloses molecular assays that involve measurement of expression level(s) of one or more genes or gene subsets from a biological sample obtained to identify a risk classification for a prostate cancer patient. For example, patients may be stratified using expression level(s) of one or more genes, positively or negatively, with positive clinical outcome of prostate cancer, or with a prognostic factor. In an exemplary embodiment, the prognostic factor is Gleason score.

The present invention provides a method of predicting the likelihood of a clinical outcome for a patient with prostate cancer comprising determination of a level of one or more RNA transcripts, or an expression product thereof, in a biological sample containing tumor cells obtained from the patient, wherein the RNA transcript, or its expression product, is selected from the 81 genes shown in FIG. 1 and listed in Tables 1A and 1B. The method comprises assigning the one or more RNA transcripts, or an expression product thereof, to one or more gene groups selected from a cellular organization gene group, basal epithelia gene group, a stress response gene group, an androgen gene group, a stromal response gene group, and a proliferation gene group. The method further comprises calculating a quantitative score for the patient by weighting the level of the one or more RNA transcripts or an expression product thereof, by their contribution to a clinical outcome and predicting the likelihood of a clinical outcome for the patient based on the quantitative score. In an embodiment of the invention, an increase in the quantitative score correlates with an increased likelihood of a negative clinical outcome.

In a particular embodiment, the one or more RNA transcripts, or an expression product thereof, is selected from BIN1, IGF1, C7, GSN, DES, TGFB1I1, TPM2, VCL, FLNC, ITGA7, COL6A1, PPP1R12A, GSTM1, GSTM2, PAGE4, PPAP2B, SRD5A2, PRKCA, IGFBP6, GPM6B, OLFML3, HLF, CYP3A5, KRT15, KRT5, LAMB3, SDC1, DUSP1, EGFR1, FOS, JUN, EGR3, GADD45B, ZFP36, FAM13C, KLK2, ASPN, SFRP4, BGN, THBS2, INHBA, COL1A1, COL3A1, COL1A2, SPARC, COL8A1, COL4A1, FN1, FAP, COL5A2, CDCl20, TPX2, UBE2T, MYBL2, and CDKN2C. BIN1, IGF1, C7, GSN, DES, TGFB1I1, TPM2, VCL, FLNC, ITGA7, COL6A1, PPP1R12A, GSTM1, GSTM2, PAGE4, PPAP2B, SRD5A2, PRKCA, IGFBP6, GPM6B, OLFML3, and HLF are assigned to the cellular organization gene group. CYP3A5, KRT15, KRT5, LAMB3, and SDC1 are assigned to the basal epithelial gene group. DUSP1, EGFR1, FOS, JUN, EGR3, GADD45B, and ZFP36 are assigned to the stress response gene group. FAM13C, KLK2, AZGP1, and SRD5A2 are assigned to the androgen gene group. ASPN, SFRP4, BGN, THBS2, INHBA, COL1A1, COL3A1, COL1A2, SPARC, COL8A1, COL4A1, FN1, FAP and COL5A2 are assigned to the stromal response gene group. CDCl20, TPX2, UBE2T, MYBL2, and CDKN2C are assigned to the proliferation gene group. The method may further comprise determining the level of at least one RNA transcript, or an expression product thereof, selected from STAT5B, NFAT5, AZGP1, ANPEP, IGFBP2, SLC22A3, ERG, AR, SRD5A2, GSTM1, and GSTM2.

In an embodiment of the invention, the level of one or more RNA transcripts, or an expression product thereof, from each of the stromal response gene group and the cellular organization gene group are determined. In another embodiment, the level of one or more RNA transcripts, or expression products thereof, from each of the stromal response gene group and PSA gene group are determined. Additionally, the level of one or more RNA transcripts, or expression products thereof, from the cellular organization gene group and/or proliferation gene group may be determined. In this embodiment, gene(s) to be assayed from the stromal response gene group may be selected from ASPN, BGN, COL1A1, SPARC, FN1, COL3A1, COL4A1, INHBA, THBS2, and SFRP4; gene(s) to be assayed from the androgen gene group may be selected from FAM13C and KLK2; gene(s) to be assayed from the cellular organization gene group may be selected from FLNC, GSN, GSTM2, IGFBP6, PPAP2B, PPP1R12A, BIN1, VCL, IGF1, TPM2, C7, and GSTM1; and gene(s) to be assayed from the proliferation gene group may be selected from TPX2, CDCl20, and MYBL2.

In a particular embodiment, the RNA transcripts, or their expression products, are selected from BGN, COL1A1, SFRP4, FLNC, GSN, TPM2, TPX2, FAM13C, KLK2, AZGP1, GSTM2, and SRD5A2. BGN, COL1A1, and SFRP4 are assigned to the stromal response gene group; FLNC, GSN, and TPM2 are assigned to the cellular organization gene group; and FAM13C and KLK2 are assigned to the androgen gene group. The level of the RNA transcripts, or their expression products, comprising at least one of the gene groups selected from the stromal response gene group, cellular organization gene group, and androgen gene group, may be determined for the method of the invention. In any of the embodiments, the androgen gene group may further comprise AZGP1 and SRD5A2.

In addition, the level of any one of the gene combinations show in Table 4 may be determined. For instance, the RS0 model in Table 4 comprises determining the levels of the RNA transcripts, or gene expression products thereof, of ASPN, BGN, COL1A1, SPARC, FLNC, GSN, GSTM2, IGFBP6, PPAP2B, PPP1R12A, TPX2, CDCl20, MYBL2, FAM13C, KLK2, STAT5B, and NFAT5. Furthermore, any one of the algorithms shown in Table 4 may be used to calculate the quantitative score for the patient.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a dendrogram depicting the association of the 81 genes selected from the gene identification study.

FIGS. 2A-2E are scatter plots showing the comparison of normalized gene expression (Cp) for matched samples from each patient where the x-axis is the gene expression from the primary Gleason pattern RP sample (PGP) and the y-axis is the gene expression from the biopsy (BX) sample. FIG. 2A: All ECM (stomal response) genes; FIG. 2B: All migration (cellular organization) genes; FIG. 2C: All proliferation genes; FIG. 2D: PSA (androgen) genes; FIG. 2E: other genes from the 81 gene list that do not fall within any of these four gene groups.

FIGS. 3A-3D are range plots of gene expression of individual genes within each gene group in the biopsy (BX) and PGP RP samples. FIG. 3A: All ECM (stromal response) genes;

FIG. 3B: All migration (cellular organization) genes; FIG. 3C: All proliferation genes; FIG. 3D: other genes from the 81 gene list that do not fall within any of the gene groups.

FIG. 4 is a schematic illustration of the clique-stack method used to identify co-expressed genes.

FIGS. 5A-5C show examples of cliques and stacks. FIG. 5A is an example of a graph that is not a clique; FIG. 5B is an example of a clique; FIG. 5C is an example of a clique but is not a maximal clique.

FIG. 6 is a graph showing two maximal cliques: 1-2-3-4-5 and 1-2-3-4-6.

FIGS. 7A-7C schematically illustrate stacking (in FIG. 7C) of two maximal cliques (shown in FIGS. 7A and 7B).

FIG. 8 is a graph showing that RS27 and CAPRA risk groups predict freedom from high-grade or non-organ-confined disease.

FIG. 9 is a graph showing that RS27 and AUA risk groups predict freedom from high-grade or non-organ-confined disease.

FIG. 10 is a graph showing time to clinical recurrence of PTEN low and PTEN normal patients from the gene identification study.

FIG. 11 is a graph showing time to clinical recurrence of patients from the gene identification study stratified into PTEN low/normal and TMPRSS-ERG negative/positive.

DEFINITIONS

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992), provide one skilled in the art with a general guide to many of the terms used in the present application.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described herein. For purposes of the invention, the following terms are defined below.

The terms “tumor” and “lesion” as used herein, refer to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. Those skilled in the art will realize that a tumor tissue sample may comprise multiple biological elements, such as one or more cancer cells, partial or fragmented cells, tumors in various stages, surrounding histologically normal-appearing tissue, and/or macro or micro-dissected tissue.

The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer in the present disclosure include cancer of the urogenital tract, such as prostate cancer.

As used herein, the term “prostate cancer” is used in the broadest sense and refers to all stages and all forms of cancer arising from the tissue of the prostate gland.

Staging of the cancer assists a physician in assessing how far the disease has progressed and to plan a treatment for the patient. Staging may be done clinically (clinical staging) by physical examination, blood tests, or response to radiation therapy, and/or pathologically (pathologic staging) based on surgery, such as radical prostatectomy. According to the tumor, node, metastasis (TNM) staging system of the American Joint Committee on Cancer (AJCC), AJCC Cancer Staging Manual (7th Ed., 2010), the various stages of prostate cancer are defined as follows: Tumor: T1: clinically inapparent tumor not palpable or visible by imaging, T1a: tumor incidental histological finding in 5% or less of tissue resected, T1b: tumor incidental histological finding in more than 5% of tissue resected, T1c: tumor identified by needle biopsy; T2: tumor confined within prostate, T2a: tumor involves one half of one lobe or less, T2b: tumor involves more than half of one lobe, but not both lobes, T2c: tumor involves both lobes; T3: tumor extends through the prostatic capsule, T3a: extracapsular extension (unilateral or bilateral), T3b: tumor invades seminal vesicle(s); T4: tumor is fixed or invades adjacent structures other than seminal vesicles (bladder neck, external sphincter, rectum, levator muscles, or pelvic wall). Generally, a clinical T (cT) stage is T1 or T2 and pathologic T (pT) stage is T2 or higher. Node: NO: no regional lymph node metastasis; N1: metastasis in regional lymph nodes. Metastasis: M0: no distant metastasis; Ml: distant metastasis present.

The Gleason Grading system is used to help evaluate the prognosis of men with prostate cancer. Together with other parameters, it is incorporated into a strategy of prostate cancer staging, which predicts prognosis and helps guide therapy. A Gleason “score” or “grade” is given to prostate cancer based upon its microscopic appearance. Tumors with a low Gleason score typically grow slowly enough that they may not pose a significant threat to the patients in their lifetimes. These patients are monitored (“watchful waiting” or “active surveillance”) over time. Cancers with a higher Gleason score are more aggressive and have a worse prognosis, and these patients are generally treated with surgery (e.g., radical prostatectomy) and, in some cases, therapy (e.g., radiation, hormone, ultrasound, chemotherapy). Gleason scores (or sums) comprise grades of the two most common tumor patterns. These patterns are referred to as Gleason patterns 1-5, with pattern 1 being the most well-differentiated. Most have a mixture of patterns. To obtain a Gleason score or grade, the dominant pattern is added to the second most prevalent pattern to obtain a number between 2 and 10. The Gleason Grades include: G1: well differentiated (slight anaplasia) (Gleason 2-4); G2: moderately differentiated (moderate anaplasia) (Gleason 5-6); G3-4: poorly differentiated/undifferentiated (marked anaplasia) (Gleason 7-10).

Stage groupings: Stage I: T1a N0 M0 G1; Stage II: (T1a N0 M0 G2-4) or (T1b, c, T1, T2, N0 M0 Any G); Stage III: T3 N0 M0 Any G; Stage IV: (T4 N0 M0 Any G) or (Any T N1 M0 Any G) or (Any T Any N M1 Any G).

The term “upgrading” as used herein refers to an increase in Gleason grade determined from biopsy to Gleason grade determined from radical prostatectomy (RP). For example, upgrading includes a change in Gleason grade from 3+3 or 3+4 on biopsy to 3+4 or greater on RP. “Significant upgrading” or “upgrade2” as used herein, refers to a change in Gleason grade from 3+3 or 3+4 determined from biopsy to 4+3 or greater, or seminal vessical involvement (SVI), or extracapsular involvement (ECE) as determined from RP.

The term “high grade” as used herein refers to Gleason score of >=3+4 or >=4+3 on RP. The term “low grade” as used herein refers to a Gleason score of 3+3 on RP. In a particular embodiment, “high grade” disease refers to Gleason score of at least major pattern 4, minor pattern 5, or tertiary pattern 5.

The term “upstaging” as used herein refers to an increase in tumor stage from biopsy to tumor stage at RP. For example, upstaging is a change in tumor stage from clinical T1 or T2 stage at biopsy to pathologic T3 stage at RP.

The term “non organ-confined disease” as used herein refers to having pathologic stage T3 disease at RP. The term “organ-confined” as used herein refers to pathologic stage pT2 at RP.

The term “adverse pathology” as used herein refers to a high grade disease as defined above, or non organ-confined disease as defined above. In a particular embodiment, “adverse pathology” refers to prostate cancer with a Gleason score of >=3+4 or >=4+3 or pathologic stage T3.

In another embodiment, the term “high-grade or non-organ-confined disease” refers to prostate cancer with a Gleason score of at least major pattern 4, minor pattern 5, or tertiary pattern 5, or pathologic stage T3.

As used herein, the terms “active surveillance” and “watchful waiting” mean closely monitoring a patient's condition without giving any treatment until symptoms appear or change. For example, in prostate cancer, watchful waiting is usually used in older men with other medical problems and early-stage disease.

As used herein, the term “surgery” applies to surgical methods undertaken for removal of cancerous tissue, including pelvic lymphadenectomy, radical prostatectomy, transurethral resection of the prostate (TURP), excision, dissection, and tumor biopsy/removal. The tumor tissue or sections used for gene expression analysis may have been obtained from any of these methods.

As used herein, the term “biological sample containing cancer cells” refers to a sample comprising tumor material obtained from a cancer patient. The term encompasses tumor tissue samples, for example, tissue obtained by radical prostatectomy and tissue obtained by biopsy, such as for example, a core biopsy or a fine needle biopsy. The biological sample may be fresh, frozen, or a fixed, wax-embedded tissue sample, such as a formalin-fixed, paraffin-embedded tissue sample. A biological sample also encompasses bodily fluids containing cancer cells, such as blood, plasma, serum, urine, and the like. Additionally, the term “biological sample containing cancer cells” encompasses a sample comprising tumor cells obtained from sites other than the primary tumor, e.g., circulating tumor cells. The term also encompasses cells that are the progeny of the patient's tumor cells, e.g. cell culture samples derived from primary tumor cells or circulating tumor cells. The term further encompasses samples that may comprise protein or nucleic acid material shed from tumor cells in vivo, e.g., bone marrow, blood, plasma, serum, and the like. The term also encompasses samples that have been enriched for tumor cells or otherwise manipulated after their procurement and samples comprising polynucleotides and/or polypeptides that are obtained from a patient's tumor material.

Prognostic factors are those variables related to the natural history of cancer that influence the recurrence rates and outcome of patients once they have developed cancer. Clinical parameters that have been associated with a worse prognosis include, for example, increased tumor stage, high PSA level at presentation, and high Gleason grade or pattern. Prognostic factors are frequently used to categorize patients into subgroups with different baseline relapse risks.

The term “prognosis” is used herein to refer to the likelihood that a cancer patient will have a cancer-attributable death or progression, including recurrence, metastatic spread, and drug resistance, of a neoplastic disease, such as prostate cancer. For example, a “good prognosis” would include long term survival without recurrence and a “bad prognosis” would include cancer recurrence.

A “positive clinical outcome” can be assessed using any endpoint indicating a benefit to the patient, including, without limitation, (1) inhibition, to some extent, of tumor growth, including slowing down and complete growth arrest; (2) reduction in the number of tumor cells; (3) reduction in tumor size; (4) inhibition (i.e., reduction, slowing down, or complete stopping) of tumor cell infiltration into adjacent peripheral organs and/or tissues; (5) inhibition of metastasis; (6) enhancement of anti-tumor immune response, possibly resulting in regression or rejection of the tumor; (7) relief, to some extent, of one or more symptoms associated with the tumor; (8) increase in the duration of survival following treatment; and/or (9) decreased mortality at a given point of time following treatment. Positive clinical outcome can also be considered in the context of an individual's outcome relative to an outcome of a population of patients having a comparable clinical diagnosis, and can be assessed using various endpoints such as an increase in the duration of Recurrence-Free Interval (RFI), an increase in survival time (Overall Survival (OS)) or prostate cancer-specific survival time (Prostate Cancer-Specific Survival (PCSS)) in a population, no upstaging or upgrading in tumor stage or Gleason grade between biopsy and radical prostatectomy, presence of 3+3 grade and organ-confined disease at radical prostatectomy, and the like.

The term “risk classification” means a grouping of subjects by the level of risk (or likelihood) that the subject will experience a particular negative clinical outcome. A subject may be classified into a risk group or classified at a level of risk based on the methods of the present disclosure, e.g. high, medium, or low risk. A “risk group” is a group of subjects or individuals with a similar level of risk for a particular clinical outcome.

The term “long-term” survival is used herein to refer to survival for a particular time period, e.g., for at least 5 years, or for at least 10 years.

The term “recurrence” is used herein to refer to local or distant recurrence (i.e., metastasis) of cancer. For example, prostate cancer can recur locally in the tissue next to the prostate or in the seminal vesicles. The cancer may also affect the surrounding lymph nodes in the pelvis or lymph nodes outside this area. Prostate cancer can also spread to tissues next to the prostate, such as pelvic muscles, bones, or other organs. Recurrence can be determined by clinical recurrence detected by, for example, imaging study or biopsy, or biochemical recurrence detected by, for example, sustained follow-up prostate-specific antigen (PSA) levels≥0.4 ng/mL or the initiation of salvage therapy as a result of a rising PSA level.

The term “clinical recurrence-free interval (cRFI)” is used herein as time from surgery to first clinical recurrence or death due to clinical recurrence of prostate cancer. If follow-up ended without occurrence of clinical recurrence, or other primary cancers or death occurred prior to clinical recurrence, time to cRFI is considered censored; when this occurs, the only information known is that up through the censoring time, clinical recurrence has not occurred in this subject. Biochemical recurrences are ignored for the purposes of calculating cRFI.

The term “biochemical recurrence-free interval (bRFI)” is used herein to mean the time from surgery to first biochemical recurrence of prostate cancer. If clinical recurrence occurred before biochemical recurrence, follow-up ended without occurrence of bRFI, or other primary cancers or death occurred prior to biochemical recurrence, time to biochemical recurrence is considered censored at the first of these.

The term “Overall Survival (OS)” is used herein to refer to the time from surgery to death from any cause. If the subject was still alive at the time of last follow-up, survival time is considered censored at the time of last follow-up. Biochemical recurrence and clinical recurrence are ignored for the purposes of calculating OS.

The term “Prostate Cancer-Specific Survival (PCSS)” is used herein to describe the time from surgery to death from prostate cancer. If the patient did not die of prostate cancer before end of followup, or died due to other causes, PCSS is considered censored at this time. Clinical recurrence and biochemical recurrence are ignored for the purposes of calculating PCSS.

In practice, the calculation of the time-to-event measures listed above may vary from study to study depending on the definition of events to be considered censored.

As used herein, the term “expression level” as applied to a gene refers to the normalized level of a gene product, e.g. the normalized value determined for the RNA level of a gene or for the polypeptide level of a gene.

The term “gene product” or “expression product” are used herein to refer to the RNA (ribonucleic acid) transcription products (transcripts) of the gene, including mRNA, and the polypeptide translation products of such RNA transcripts. A gene product can be, for example, an unspliced RNA, an mRNA, a splice variant mRNA, a microRNA, a fragmented RNA, a polypeptide, a post-translationally modified polypeptide, a splice variant polypeptide, etc.

The term “RNA transcript” as used herein refers to the RNA transcription products of a gene, including, for example, mRNA, an unspliced RNA, a splice variant mRNA, a microRNA, and a fragmented RNA.

Unless indicated otherwise, each gene name used herein corresponds to the Official Symbol assigned to the gene and provided by Entrez Gene (URL: www.ncbi.nlm.nih.gov/sites/entrez) as of the filing date of this application.

The term “microarray” refers to an ordered arrangement of hybridizable array elements, e.g. oligonucleotide or polynucleotide probes, on a substrate.

The term “polynucleotide” generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotides as defined herein include, without limitation, single- and double-stranded DNA, DNA including single- and double-stranded regions, single- and double-stranded RNA, and RNA including single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or include single- and double-stranded regions. In addition, the term “polynucleotide” as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. The term “polynucleotide” specifically includes cDNAs. The term includes DNAs (including cDNAs) and RNAs that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons, are “polynucleotides” as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritiated bases, are included within the term “polynucleotides” as defined herein. In general, the term “polynucleotide” embraces all chemically, enzymatically and/or metabolically modified forms of unmodified polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells.

The term “oligonucleotide” refers to a relatively short polynucleotide, including, without limitation, single-stranded deoxyribonucleotides, single- or double-stranded ribonucleotides, RNArDNA hybrids and double-stranded DNAs. Oligonucleotides, such as single-stranded DNA probe oligonucleotides, are often synthesized by chemical methods, for example using automated oligonucleotide synthesizers that are commercially available. However, oligonucleotides can be made by a variety of other methods, including in vitro recombinant DNA-mediated techniques and by expression of DNAs in cells and organisms.

The term “Ct” as used herein refers to threshold cycle, the cycle number in quantitative polymerase chain reaction (qPCR) at which the fluorescence generated within a reaction well exceeds the defined threshold, i.e. the point during the reaction at which a sufficient number of amplicons have accumulated to meet the defined threshold.

The term “Cp” as used herein refers to “crossing point.” The Cp value is calculated by determining the second derivatives of entire qPCR amplification curves and their maximum value. The Cp value represents the cycle at which the increase of fluorescence is highest and where the logarithmic phase of a PCR begins.

The terms “threshold” or “thresholding” refer to a procedure used to account for non-linear relationships between gene expression measurements and clinical response as well as to further reduce variation in reported patient scores. When thresholding is applied, all measurements below or above a threshold are set to that threshold value. A non-linear relationship between gene expression and outcome could be examined using smoothers or cubic splines to model gene expression on recurrence free interval using Cox PH regression or on adverse pathology status using logistic regression. D. Cox, Journal of the Royal Statistical Society, Series B 34:187-220 (1972). Variation in reported patient scores could be examined as a function of variability in gene expression at the limit of quantitation and/or detection for a particular gene.

As used herein, the term “amplicon,” refers to pieces of DNA that have been synthesized using amplification techniques, such as polymerase chain reactions (PCR) and ligase chain reactions.

“Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to re-anneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology (Wiley Interscience Publishers, 1995).

“Stringent conditions” or “high stringency conditions”, as defined herein, typically: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodium citrate) and 50% formamide, followed by a high-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and % SDS) less stringent that those described above. An example of moderately stringent conditions is overnight incubation at 37° C. in a solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1×SSC at about 37-500 C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.

The terms “splicing” and “RNA splicing” are used interchangeably and refer to RNA processing that removes introns and joins exons to produce mature mRNA with continuous coding sequence that moves into the cytoplasm of an eukaryotic cell.

As used herein, the term “TMPRSS fusion” and “TMPRSS2 fusion” are used interchangeably and refer to a fusion of the androgen-driven TMPRSS2 gene with the ERG oncogene, which has been demonstrated to have a significant association with prostate cancer. S. Perner, et al., Urologe A. 46(7):754-760 (2007); S. A. Narod, et al., Br J Cancer 99(6):847-851 (2008). As used herein, positive TMPRSS fusion status indicates that the TMPRSS fusion is present in a tissue sample, whereas negative TMPRSS fusion status indicates that the TMPRSS fusion is not present in a tissue sample. Experts skilled in the art will recognize that there are numerous ways to determine TMPRSS fusion status, such as real-time, quantitative PCR or high-throughput sequencing. See, e.g., K. Mertz, et al., Neoplasis 9(3):200-206 (2007); C. Maher, Nature 458(7234):97-101 (2009).

The terms “correlated” and “associated” are used interchangeably herein to refer to the association between two measurements (or measured entities). The disclosure provides genes or gene subsets, the expression levels of which are associated with clinical outcome. For example, the increased expression level of a gene may be positively correlated (positively associated) with a good or positive clinical outcome. Such a positive correlation may be demonstrated statistically in various ways, e.g. by a cancer recurrence hazard ratio less than one or by a cancer upgrading or upstaging odds ratio of less than one. In another example, the increased expression level of a gene may be negatively correlated (negatively associated) with a good or positive clinical outcome. In that case, for example, the patient may experience a cancer recurrence or upgrading/upstaging of the cancer, and this may be demonstrated statistically in various ways, e.g., a hazard ratio greater than 1 or an odds ratio greater than one. “Correlation” is also used herein to refer to the strength of association between the expression levels of two different genes, such that the expression level of a first gene can be substituted with an expression level of a second gene in a given algorithm if their expression levels are highly correlated. Such “correlated expression” of two genes that are substitutable in an algorithm are usually gene expression levels that are positively correlated with one another, e.g., if increased expression of a first gene is positively correlated with an outcome (e.g., increased likelihood of good clinical outcome), then the second gene that is co-expressed and exhibits correlated expression with the first gene is also positively correlated with the same outcome.

The terms “co-express” and “co-expressed”, as used herein, refer to a statistical correlation between the amounts of different transcript sequences across a population of different patients. Pairwise co-expression may be calculated by various methods known in the art, e.g., by calculating Pearson correlation coefficients or Spearman correlation coefficients. Co-expressed gene cliques may also be identified by seeding and stacking the maximal clique enumeration (MCE) described in Example 4 herein. An analysis of co-expression may be calculated using normalized expression data. Genes within the same gene subset are also considered to be co-expressed.

A “computer-based system” refers to a system of hardware, software, and data storage medium used to analyze information. The minimum hardware of a patient computer-based system comprises a central processing unit (CPU), and hardware for data input, data output (e.g., display), and data storage. An ordinarily skilled artisan can readily appreciate that any currently available computer-based systems and/or components thereof are suitable for use in connection with the methods of the present disclosure. The data storage medium may comprise any manufacture comprising a recording of the present information as described above, or a memory access device that can access such a manufacture.

To “record” data, programming or other information on a computer readable medium refers to a process for storing information, using any such methods as known in the art. Any convenient data storage structure may be chosen, based on the means used to access the stored information. A variety of data processor programs and formats can be used for storage, e.g. word processing text file, database format, etc.

A “processor” or “computing means” references any hardware and/or software combination that will perform the functions required of it. For example, a suitable processor may be a programmable digital microprocessor such as available in the form of an electronic controller, mainframe, server or personal computer (desktop or portable). Where the processor is programmable, suitable programming can be communicated from a remote location to the processor, or previously saved in a computer program product (such as a portable or fixed computer readable storage medium, whether magnetic, optical or solid state device based). For example, a magnetic medium or optical disk may carry the programming, and can be read by a suitable reader communicating with each processor at its corresponding station.

Algorithm-Based Methods and Gene Subsets

The present invention provides an algorithm-based molecular diagnostic assay for predicting a clinical outcome for a patient with prostate cancer. The expression level of one or more genes may be used alone or arranged into functional gene subsets to calculate a quantitative score that can be used to predict the likelihood of a clinical outcome. The algorithm-based assay and associated information provided by the practice of the methods of the present invention facilitate optimal treatment decision-making in prostate cancer. For example, such a clinical tool would enable physicians to identify patients who have a low likelihood of having an aggressive cancer and therefore would not need RP, or who have a high likelihood of having an aggressive cancer and therefore would need RP.

As used herein, a “quantitative score” is an arithmetically or mathematically calculated numerical value for aiding in simplifying or disclosing or informing the analysis of more complex quantitative information, such as the correlation of certain expression levels of the disclosed genes or gene subsets to a likelihood of a clinical outcome of a prostate cancer patient. A quantitative score may be determined by the application of a specific algorithm. The algorithm used to calculate the quantitative score in the methods disclosed herein may group the expression level values of genes. The grouping of genes may be performed at least in part based on knowledge of the relative contribution of the genes according to physiologic functions or component cellular characteristics, such as in the groups discussed herein. A quantitative score may be determined for a gene group (“gene group score”). The formation of groups, in addition, can facilitate the mathematical weighting of the contribution of various expression levels of genes or gene subsets to the quantitative score. The weighting of a gene or gene group representing a physiological process or component cellular characteristic can reflect the contribution of that process or characteristic to the pathology of the cancer and clinical outcome, such as recurrence or upgrading/upstaging of the cancer. The present invention provides a number of algorithms for calculating the quantitative scores, for example, as set forth in Table 4. In an embodiment of the invention, an increase in the quantitative score indicates an increased likelihood of a negative clinical outcome.

In an embodiment, a quantitative score is a “recurrence score,” which indicates the likelihood of a cancer recurrence, upgrading or upstaging of a cancer, adverse pathology, non-organ-confined disease, high-grade disease, and/or highgrade or non-organ-confined disease. An increase in the recurrence score may correlate with an increase in the likelihood of cancer recurrence, upgrading or upstaging of a cancer, adverse pathology, non-organ-confined disease, high-grade disease, and/or highgrade or non-organ-confined disease.

The gene subsets of the present invention include an ECM gene group, migration gene group, androgen gene group, proliferation gene group, epithelia gene group, and stress gene group.

The gene subsets referred to herein as the “ECM gene group,” “stromal gene group,” and “stromal response gene group” are used interchangeably and include genes that are synthesized predominantly by stromal cells and are involved in stromal response and genes that co-express with the genes of the ECM gene group. “Stromal cells” are referred to herein as connective tissue cells that make up the support structure of biological tissues. Stromal cells include fibroblasts, immune cells, pericytes, endothelial cells, and inflammatory cells. “Stromal response” refers to a desmoplastic response of the host tissues at the site of a primary tumor or invasion. See, e.g., E. Rubin, J. Farber, Pathlogy, 985-986 (end Ed. 1994). The ECM gene group includes, for example, ASPN, SFRP4, BGN, THBS2, INHBA, COL1A1, COL3A1, COL1A2, SPARC, COL8A1, COL4A1, FN1, FAP, and COL5A2, and co-expressed genes thereof. Exemplary co-expressed genes include the genes and/or gene cliques shown in Table 8.

The gene subsets referred to herein as the “migration gene group” or “migration regulation gene group” or “cytoskeletal gene group” or “cellular organization gene group” are used interchangeably and include genes and co-expressed genes that are part of a dynamic microfilament network of actin and accessory proteins and that provide intracellular support to cells, generate the physical forces for cell movement and cell division, as well as facilitate intracellular transport of vesicles and cellular organelle. The migration gene group includes, for example, BIN1, IGF1, C7, GSN, DES, TGFB1I1, TPM2, VCL, FLNC, ITGA7, COL6A1, PPP1R12A, GSTM1, GSTM2, PAGE4, PPAP2B, SRD5A2, PRKCA, IGFBP6, GPM6B, OLFML3, and HLF, and co-expressed genes thereof. Exemplary co-expressed genes and/or gene cliques are provided in Table 9.

The gene subset referred to herein as the “androgen gene group,” “PSA gene group,” and “PSA regulation gene group” are used interchangeably and include genes that are members of the kallikrein family of serine proteases (e.g. kallikrein 3 [PSA]), and genes that co-express with genes of the androgen gene group. The androgen gene group includes, for example, FAM13C and KLK2, and co-expressed genes thereof. The androgen gene group may further comprise AZGP1 and SRD5A2, and co-expressed genes thereof.

The gene subsets referred to herein as the “proliferation gene group” and “cell cycle gene group” are used interchangeably and include genes that are involved with cell cycle functions and genes that co-express with genes of the proliferation gene group. “Cell cycle functions” as used herein refers to cell proliferation and cell cycle control, e.g., checkpoint/G1 to S phase transition. The proliferation gene group thus includes, for example, CDCl20, TPX2, UBE2T, MYBL2, and CDKN2C, and co-expressed genes thereof. Exemplary co-expressed genes and/or gene cliques are provided in Table 10.

The gene subsets referred to herein as the “epithelia gene group” and “basal epithelia gene group” are used interchangeably and include genes that are expressed during the differentiation of a polarized epithelium and that provide intracellular structural integrity to facilitate physical interactions with neighboring epithelial cells, and genes that co-express with genes of the epithelia gene group. The epithelia gene group includes, for example, CYP3A5, KRT15, KRT5, LAMB3, and SDC1 and co-expressed genes thereof.

The gene subset referred to herein as the “stress gene group,” “stress response gene group,” and “early response gene group” are used interchangeably and includes genes and co-expressed genes that are transcription factors and DNA-binding proteins activated rapidly and transiently in response to cellular stress and other extracellular signals. These factors, in turn, regulate the transcription of a diverse range of genes. The stress gene group includes, for example, DUSP1, EGR1, FOS, JUN, EGR3, GADD45B, and ZFP36, and co-expressed genes thereof. Exemplary co-expressed genes and/or gene cliques are provided in Table 11.

Expression levels of other genes and their co-expressed genes may be used with one more of the above gene subsets to predict a likelihood of a clinical outcome of a prostate cancer patient. For example, the expression level of one or more genes selected from the 81 genes of FIG. 1 or Table 1A or 1B that do not fall within any of the disclosed gene subsets may be used with one or more of the disclosed gene subsets. In an embodiment of the invention, one or more of STAT5B, NFAT5, AZGP1, ANPEP, IGFBP2, SLC22A3, ERG, AR, SRD5A2, GSTM1, and GSTM2 may be used in one or more gene subsets described above to predict a likelihood of a clinical outcome.

The present invention also provides methods to determine a threshold expression level for a particular gene. A threshold expression level may be calculated for a specific gene. A threshold expression level for a gene may be based on a normalized expression level. In one example, a C_(p)threshold expression level may be calculated by assessing functional forms using logistic regression or Cox proportional hazards regression.

The present invention further provides methods to determine genes that co-express with particular genes identified by, e.g., quantitative RT-PCR (qRT-PCR), as validated biomarkers relevant to a particular type of cancer. The co-expressed genes are themselves useful biomarkers. The co-expressed genes may be substituted for the genes with which they co-express. The methods can include identifying gene cliques from microarray data, normalizing the microarray data, computing a pairwise Spearman correlation matrix for the array probes, filtering out significant co-expressed probes across different studies, building a graph, mapping the probe to genes, and generating a gene clique report. An exemplary method for identifying co-expressed genes is described in Example 3 below, and co-expressed genes identified using this method are provided in Tables 8-11. The expression levels of one or more genes of a gene clique may be used to calculate the likelihood that a patient with prostate cancer will experience a positive clinical outcome, such as a reduced likelihood of a cancer recurrence.

Any one or more combinations of gene groups may be assayed in the method of the present invention. For example, a stromal response gene group may be assayed, alone or in combination, with a cellular organization gene group, a proliferation gene group, and/or an androgen gene group. In addition, any number of genes within each gene group may be assayed.

In a specific embodiment of the invention, a method for predicting a clinical outcome for a patient with prostate cancer comprises measuring an expression level of at least one gene from a stromal response gene group, or a co-expressed gene thereof, and at least one gene from a cellular organization gene group, or a co-expressed gene thereof. In another embodiment, the expression level of at least two genes from a stromal response gene group, or a co-expressed gene thereof, and at least two genes from a cellular organization gene group, or a co-expressed gene thereof, are measured. In yet another embodiment, the expression levels of at least three genes are measured from each of the stromal response gene group and the cellular organization gene group. In a further embodiment, the expression levels of at least four genes are measured from each of the stromal response gene group and the cellular organization gene group. In another embodiment, the expression levels of at least five genes are measured from each of the stromal response gene group and the cellular organization gene group. In yet a further embodiment, the expression levels of at least six genes are measured from each of the stromal response gene group and the cellular organization gene group.

In another specific embodiment, the expression level of at least one gene from the stromal response gene group, or a co-expressed gene thereof, may be measured in addition to the expression level of at least one gene from an androgen gene group, or a co-expressed gene thereof. In a particular embodiment, the expression levels of at least three genes, or co-expressed genes thereof, from the stromal response gene group, and the expression level of at least one gene, or co-expressed gene thereof, from the androgen gene group may be measured.

In a further embodiment, the expression level of at least one gene each from the stromal response gene group, the androgen gene group, and the cellular organization gene group, or co-expressed genes thereof, may be measured. In a particular embodiment, the level of at least three genes from the stromal resonse gene group, at least one gene from the androgen gene group, and at least three genes from the cellular organization gene group may be measured. In another embodiment, the expression level of at least one gene each from the stromal response gene group, the androgen gene group, and the proliferation gene group, or co-expressed genes thereof, may be measured. In a particular embodiment, the level of at least three genes from the stromal response gene group, at least one gene from the androgen gene group, and at least one gene from the proliferation gene group may be measured. In either of these combinations, at least two genes from the androgen gene group may also be measured. In any of the combinations, at least four genes from the androgen gene group may also be measured.

In another embodiment, the expression level of at least one gene each from the stromal response gene group, the androgen gene group, the cellular organization gene group, and the proliferation gene group, or co-expressed genes thereof, may be measured. In a particular embodiment, the level of at least three genes from the stromal response gene group, at least three genes from the cellular organization gene group, at least one gene from the proliferation gene group, and at least two genes from the androgen gene group may be measured. In any of the embodiments, at least four genes from the androgen gene group may be measured.

Additionally, expression levels of one or more genes that do not fall within the gene subsets described herein may be measured with any of the combinations of the gene subsets described herein. Alternatively, any gene that falls within a gene subset may be analyzed separately from the gene subset, or in another gene subset. For example, the expression levels of at least one, at least two, at least three, or at least 4 genes may be measured in addition to the gene subsets described herein. In an embodiment of the invention, the additional gene(s) are selected from STAT5B, NFAT5, AZGP1, ANPEP, IGFBP2, SLC22A3, ERG, AR, SRD5A2, GSTM1, and GSTM2.

In a specific embodiment, the method of the invention comprises measuring the expression levels of the specific combinations of genes and gene subsets shown in Table 4. In a further embodiment, gene group score(s) and quantitative score(s) are calculated according to the algorithm(s) shown in Table 4.

Various technological approaches for determination of expression levels of the disclosed genes are set forth in this specification, including, without limitation, RT-PCR, microarrays, high-throughput sequencing, serial analysis of gene expression (SAGE) and Digital Gene Expression (DGE), which will be discussed in detail below. In particular aspects, the expression level of each gene may be determined in relation to various features of the expression products of the gene including exons, introns, protein epitopes and protein activity.

The expression product that is assayed can be, for example, RNA or a polypeptide. The expression product may be fragmented. For example, the assay may use primers that are complementary to target sequences of an expression product and could thus measure full transcripts as well as those fragmented expression products containing the target sequence. Further information is provided in Table A.

The RNA expression product may be assayed directly or by detection of a cDNA product resulting from a PCR-based amplification method, e.g., quantitative reverse transcription polymerase chain reaction (qRT-PCR). (See e.g., U.S. Pat. No. 7,587,279). Polypeptide expression product may be assayed using immunohistochemistry (IHC) by proteomics techniques. Further, both RNA and polypeptide expression products may also be assayed using microarrays.

Methods of Assaying Expression Levels of a Gene Product

Methods of gene expression profiling include methods based on hybridization analysis of polynucleotides, methods based on sequencing of polynucleotides, and proteomics-based methods. Exemplary methods known in the art for the quantification of RNA expression in a sample include northern blotting and in situ hybridization (Parker & Barnes, Methods in Molecular Biology 106:247-283 (1999)); RNAse protection assays (Hod, Biotechniques 13:852-854 (1992)); and PCR-based methods, such as reverse transcription PCR (RT-PCR) (Weis et al., Trends in Genetics 8:263-264 (1992)). Antibodies may be employed that can recognize sequence-specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Representative methods for sequencing-based gene expression analysis include Serial Analysis of Gene Expression (SAGE), and gene expression analysis by massively parallel signature sequencing (MPSS). Other methods known in the art may be used.

Reverse Transcription PCR (RT-PCR)

Typically, mRNA is isolated from a test sample. The starting material is typically total RNA isolated from a human tumor, usually from a primary tumor. Optionally, normal tissues from the same patient can be used as an internal control. Such normal tissue can be histologically-appearing normal tissue adjacent to a tumor. mRNA can be extracted from a tissue sample, e.g., from a sample that is fresh, frozen (e.g. fresh frozen), or paraffin-embedded and fixed (e.g. formalin-fixed).

General methods for mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997). Methods for RNA extraction from paraffin embedded tissues are disclosed, for example, in Rupp and Locker, Lab Invest. 56:A67 (1987), and De Andres et al., BioTechniques 18:42044 (1995). In particular, RNA isolation can be performed using a purification kit, buffer set and protease from commercial manufacturers, such as Qiagen, according to the manufacturer's instructions. For example, total RNA from cells in culture can be isolated using Qiagen RNeasy mini-columns. Other commercially available RNA isolation kits include MasterPure™ Complete DNA and RNA Purification Kit (EPICENTRE®, Madison, Wis.), and Paraffin Block RNA Isolation Kit (Ambion, Inc.). Total RNA from tissue samples can be isolated using RNA Stat-60 (Tel-Test). RNA prepared from tumor can be isolated, for example, by cesium chloride density gradient centrifugation.

The sample containing the RNA is then subjected to reverse transcription to produce cDNA from the RNA template, followed by exponential amplification in a PCR reaction. The two most commonly used reverse transcriptases are avilo myeloblastosis virus reverse transcriptase (AMV-RT) and Moloney murine leukemia virus reverse transcriptase (MMLV-RT). The reverse transcription step is typically primed using specific primers, random hexamers, or oligo-dT primers, depending on the circumstances and the goal of expression profiling. For example, extracted RNA can be reverse-transcribed using a GeneAmp RNA PCR kit (Perkin Elmer, CA, USA), following the manufacturer's instructions. The derived cDNA can then be used as a template in the subsequent PCR reaction.

PCR-based methods use a thermostable DNA-dependent DNA polymerase, such as a Taq DNA polymerase. For example, TaqMan® PCR typically utilizes the 5′-nuclease activity of Taq or Tth polymerase to hydrolyze a hybridization probe bound to its target amplicon, but any enzyme with equivalent 5′ nuclease activity can be used. Two oligonucleotide primers are used to generate an amplicon typical of a PCR reaction product. A third oligonucleotide, or probe, can be designed to facilitate detection of a nucleotide sequence of the amplicon located between the hybridization sites the two PCR primers. The probe can be detectably labeled, e.g., with a reporter dye, and can further be provided with both a fluorescent dye, and a quencher fluorescent dye, as in a Taqman® probe configuration. Where a Taqman® probe is used, during the amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner. The resultant probe fragments disassociate in solution, and signal from the released reporter dye is free from the quenching effect of the second fluorophore. One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.

TaqMan® RT-PCR can be performed using commercially available equipment, such as, for example, high-throughput platforms such as the ABI PRISM 7700 Sequence Detection System® (Perkin-Elmer-Applied Biosystems, Foster City, Calif., USA), or Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany). In a preferred embodiment, the procedure is run on a LightCycler® 480 (Roche Diagnostics) real-time PCR system, which is a microwell plate-based cycler platform.

5′-Nuclease assay data are commonly initially expressed as a threshold cycle (“Ct”). Fluorescence values are recorded during every cycle and represent the amount of product amplified to that point in the amplification reaction. The threshold cycle (Ct) is generally described as the point when the fluorescent signal is first recorded as statistically significant. Alternatively, data may be expressed as a crossing point (“Cp”). The Cp value is calculated by determining the second derivatives of entire qPCR amplification curves and their maximum value. The Cp value represents the cycle at which the increase of fluorescence is highest and where the logarithmic phase of a PCR begins.

To minimize errors and the effect of sample-to-sample variation, RT-PCR is usually performed using an internal standard. The ideal internal standard gene (also referred to as a reference gene) is expressed at a quite constant level among cancerous and non-cancerous tissue of the same origin (i.e., a level that is not significantly different among normal and cancerous tissues), and is not significantly affected by the experimental treatment (i.e., does not exhibit a significant difference in expression level in the relevant tissue as a result of exposure to chemotherapy), and expressed at a quite constant level among the same tissue taken from different patients. For example, reference genes useful in the methods disclosed herein should not exhibit significantly different expression levels in cancerous prostate as compared to normal prostate tissue. Exemplary reference genes used for normalization comprise one or more of the following genes: AAMP, ARF1, ATP5E, CLTC, GPS1, and PGK1. Gene expression measurements can be normalized relative to the mean of one or more (e.g., 2, 3, 4, 5, or more) reference genes. Reference-normalized expression measurements can range from 2 to 15, where a one unit increase generally reflects a 2-fold increase in RNA quantity.

Real time PCR is compatible both with quantitative competitive PCR, where an internal competitor for each target sequence is used for normalization, and with quantitative comparative PCR using a normalization gene contained within the sample, or a housekeeping gene for RT-PCR. For further details see, e.g. Held et al., Genome Research 6:986-994 (1996).

The steps of a representative protocol for use in the methods of the present disclosure use fixed, paraffin-embedded tissues as the RNA source. For example, mRNA isolation, purification, primer extension and amplification can be performed according to methods available in the art. (see, e.g., Godfrey et al. J. Molec. Diagnostics 2: 84-91 (2000); Specht et al., Am. J. Pathol. 158: 419-29 (2001)). Briefly, a representative process starts with cutting about 10 μm thick sections of paraffin-embedded tumor tissue samples. The RNA is then extracted, and protein and DNA depleted from the RNA-containing sample. After analysis of the RNA concentration, RNA is reverse transcribed using gene-specific primers followed by RT-PCR to provide for cDNA amplification products.

Design of Intron-Based PCR Primers and Probes

PCR primers and probes can be designed based upon exon or intron sequences present in the mRNA transcript of the gene of interest. Primer/probe design can be performed using publicly available software, such as the DNA BLAT software developed by Kent, W. J., Genome Res. 12(4):656-64 (2002), or by the BLAST software including its variations.

Where necessary or desired, repetitive sequences of the target sequence can be masked to mitigate non-specific signals. Exemplary tools to accomplish this include the Repeat Masker program available on-line through the Baylor College of Medicine, which screens DNA sequences against a library of repetitive elements and returns a query sequence in which the repetitive elements are masked. The masked intron sequences can then be used to design primer and probe sequences using any commercially or otherwise publicly available primer/probe design packages, such as Primer Express (Applied Biosystems); MGB assay-by-design (Applied Biosystems); Primer3 (Steve Rozen and Helen J. Skaletsky (2000) Primer3 on the WWW for general users and for biologist programmers. See S. Rrawetz, S. Misener, Bioinformatics Methods and Protocols: Methods in Molecular Biology, pp. 365-386 (Humana Press).

Other factors that can influence PCR primer design include primer length, melting temperature (Tm), and G/C content, specificity, complementary primer sequences, and 3′-end sequence. In general, optimal PCR primers are generally 17-30 bases in length, and contain about 20-80%, such as, for example, about 50-60% G+C bases, and exhibit Tm's between 50 and 80 OC, e.g. about 50 to 70° C.

For further guidelines for PCR primer and probe design see, e.g. Dieffenbach, C W. et al, “General Concepts for PCR Primer Design” in: PCR Primer, A Laboratory Manual, Cold Spring Harbor Laboratory Press. New York, 1995, pp. 133-155; Innis and Gelfand, “Optimization of PCRs” in: PCR Protocols, A Guide to Methods and Applications, CRC Press, London, 1994, pp. 5-11; and Plasterer, T. N. Primerselect: Primer and probe design. Methods MoI. Biol. 70:520-527 (1997), the entire disclosures of which are hereby expressly incorporated by reference.

Table A provides further information concerning the primer, probe, and amplicon sequences associated with the Examples disclosed herein.

MassARRAY® System

In MassARRAY-based methods, such as the exemplary method developed by Sequenom, Inc. (San Diego, Calif.) following the isolation of RNA and reverse transcription, the obtained cDNA is spiked with a synthetic DNA molecule (competitor), which matches the targeted cDNA region in all positions, except a single base, and serves as an internal standard. The cDNA/competitor mixture is PCR amplified and is subjected to a post-PCR shrimp alkaline phosphatase (SAP) enzyme treatment, which results in the dephosphorylation of the remaining nucleotides. After inactivarion of the alkaline phosphatase, the PCR products from the competitor and cDNA are subjected to primer extension, which generates distinct mass signals for the competitor- and cDNA-derives PCR products. After purification, these products are dispensed on a chip array, which is pre-loaded with components needed for analysis with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. The cDNA present in the reaction is then quantified by analyzing the ratios of the peak areas in the mass spectrum generated. For further details see, e.g. Ding and Cantor, Proc. Natl. Acad. Sci. USA 100:3059-3064 (2003).

Other PCR-Based Methods

Further PCR-based techniques that can find use in the methods disclosed herein include, for example, BeadArray® technology (Illumina, San Diego, Calif.; Oliphant et al., Discovery of Markers for Disease (Supplement to Biotechniques), June 2002; Ferguson et al., Analytical Chemistry 72:5618 (2000)); BeadsArray for Detection of Gene Expression® (BADGE), using the commercially available LuminexlOO LabMAP® system and multiple color-coded microspheres (Luminex Corp., Austin, Tex.) in a rapid assay for gene expression (Yang et al., Genome Res. 11:1888-1898 (2001)); and high coverage expression profiling (HiCEP) analysis (Fukumura et al., Nucl. Acids. Res. 31(16) e94 (2003).

Microarrays

Expression levels of a gene or microArray of interest can also be assessed using the microarray technique. In this method, polynucleotide sequences of interest (including cDNAs and oligonucleotides) are arrayed on a substrate. The arrayed sequences are then contacted under conditions suitable for specific hybridization with detectably labeled cDNA generated from RNA of a test sample. As in the RT-PCR method, the source of RNA typically is total RNA isolated from a tumor sample, and optionally from normal tissue of the same patient as an internal control or cell lines. RNA can be extracted, for example, from frozen or archived paraffin-embedded and fixed (e.g. formalin-fixed) tissue samples.

For example, PCR amplified inserts of cDNA clones of a gene to be assayed are applied to a substrate in a dense array. Usually at least 10,000 nucleotide sequences are applied to the substrate. For example, the microarrayed genes, immobilized on the microchip at 10,000 elements each, are suitable for hybridization under stringent conditions. Fluorescently labeled cDNA probes may be generated through incorporation of fluorescent nucleotides by reverse transcription of RNA extracted from tissues of interest. Labeled cDNA probes applied to the chip hybridize with specificity to each spot of DNA on the array. After washing under stringent conditions to remove non-specifically bound probes, the chip is scanned by confocal laser microscopy or by another detection method, such as a CCD camera. Quantitation of hybridization of each arrayed element allows for assessment of corresponding RNA abundance.

With dual color fluorescence, separately labeled cDNA probes generated from two sources of RNA are hybridized pair wise to the array. The relative abundance of the transcripts from the two sources corresponding to each specified gene is thus determined simultaneously. The miniaturized scale of the hybridization affords a convenient and rapid evaluation of the expression pattern for large numbers of genes. Such methods have been shown to have the sensitivity required to detect rare transcripts, which are expressed at a few copies per cell, and to reproducibly detect at least approximately two-fold differences in the expression levels (Schena et at, Proc. Natl. Acad. ScL USA 93(2):106-149 (1996)). Microarray analysis can be performed by commercially available equipment, following manufacturer's protocols, such as by using the Affymetrix GenChip® technology, or Incyte's microarray technology.

Serial Analysis of Gene Expression (SAGE)

Serial analysis of gene expression (SAGE) is a method that allows the simultaneous and quantitative analysis of a large number of gene transcripts, without the need of providing an individual hybridization probe for each transcript. First, a short sequence tag (about 10-14 bp) is generated that contains sufficient information to uniquely identify a transcript, provided that the tag is obtained from a unique position within each transcript. Then, many transcripts are linked together to form long serial molecules, that can be sequenced, revealing the identity of the multiple tags simultaneously. The expression pattern of any population of transcripts can be quantitatively evaluated by determining the abundance of individual tags, and identifying the gene corresponding to each tag. For more details see, e.g. Velculescu et al., Science 270:484-487 (1995); and Velculescu et al., Cell 88:243-51 (1997).

Gene Expression Analysis by Nucleic Acid Sequencing

Nucleic acid sequencing technologies are suitable methods for analysis of gene expression. The principle underlying these methods is that the number of times a cDNA sequence is detected in a sample is directly related to the relative expression of the RNA corresponding to that sequence. These methods are sometimes referred to by the term Digital Gene Expression (DGE) to reflect the discrete numeric property of the resulting data. Early methods applying this principle were Serial Analysis of Gene Expression (SAGE) and Massively Parallel Signature Sequencing (MPSS). See, e.g., S. Brenner, et al., Nature Biotechnology 18(6):630-634 (2000). More recently, the advent of “next-generation” sequencing technologies has made DGE simpler, higher throughput, and more affordable. As a result, more laboratories are able to utilize DGE to screen the expression of more genes in more individual patient samples than previously possible. See, e.g., J. Marioni, Genome Research 18(9):1509-1517 (2008); R. Morin, Genome Research 18(4):610-621 (2008); A. Mortazavi, Nature Methods 5(7):621-628 (2008); N. Cloonan, Nature Methods 5(7):613-619 (2008).

Isolating RNA from Body Fluids

Methods of isolating RNA for expression analysis from blood, plasma and serum (see, e.g., K. Enders, et al., Clin Chem 48, 1647-53 (2002) (and references cited therein) and from urine (see, e.g., R. Boom, et al., J Clin Microbiol. 28, 495-503 (1990) and references cited therein) have been described.

Immunohistochemistry

Immunohistochemistry methods are also suitable for detecting the expression levels of genes and applied to the method disclosed herein. Antibodies (e.g., monoclonal antibodies) that specifically bind a gene product of a gene of interest can be used in such methods. The antibodies can be detected by direct labeling of the antibodies themselves, for example, with radioactive labels, fluorescent labels, hapten' labels such as, biotin, or an enzyme such as horse radish peroxidase or alkaline phosphatase. Alternatively, unlabeled primary antibody can be used in conjunction with a labeled secondary antibody specific for the primary antibody. Immunohistochemistry protocols and kits are well known in the art and are commercially available.

Proteomics

The term “proteome” is defined as the totality of the proteins present in a sample (e.g. tissue, organism, or cell culture) at a certain point of time. Proteomics includes, among other things, study of the global changes of protein expression in a sample (also referred to as “expression proteomics”). Proteomics typically includes the following steps: (1) separation of individual proteins in a sample by 2-D gel electrophoresis (2-D PAGE); (2) identification of the individual proteins recovered from the gel, e.g. my mass spectrometry or N-terminal sequencing, and (3) analysis of the data using bioinformatics.

General Description of the mRNA Isolation, Purification and Amplification

The steps of a representative protocol for profiling gene expression using fixed, paraffin-embedded tissues as the RNA source, including mRNA isolation, purification, primer extension and amplification are provided in various published journal articles. (See, e.g., T. E. Godfrey, et al. J. Molec. Diagnostics 2: 84-91 (2000); K. Specht et al., Am. J. Pathol. 158: 419-29 (2001), M. Cronin, et al., Am J Pathol 164:35-42 (2004)). Briefly, a representative process starts with cutting a tissue sample section (e.g. about 10 μm thick sections of a paraffin-embedded tumor tissue sample). The RNA is then extracted, and protein and DNA are removed. After analysis of the RNA concentration, RNA repair is performed if desired. The sample can then be subjected to analysis, e.g., by reverse transcribed using gene specific promoters followed by RT-PCR.

Statistical Analysis of Expression Levels in Identification of Genes

One skilled in the art will recognize that there are many statistical methods that may be used to determine whether there is a significant relationship between a clinical outcome of interest (e.g., recurrence) and expression levels of a marker gene as described here. In an exemplary embodiment, the present invention includes three studies. The first study is a stratified cohort sampling design (a form of case-control sampling) using tissue and data from prostate cancer patients. Selection of specimens was stratified by clinical T-stage (T1, T2), year of surgery (<1993, ≥1993), and prostatectomy Gleason Score (low/intermediate, high). All patients with clinical recurrence were selected and a stratified random sample of patients who did not experience a clinical recurrence was selected. For each patient, up to two enriched tumor specimens and one normal-appearing tissue sample were assayed. The second study used a subset of 70 patients from the first study from whom matched prostate biopsy tumor tissue was assayed. The third study includes all patients (170 evaluable patients) who had surgery for their prostate cancer between 1999 and 2010 at the Cleveland Clinic (CC) and had Low or Intermediate risk (by AUA) clinically localized prostate cancer who might have been reasonable candidates for active surveillance but who underwent RP at CC within 6 months of the diagnosis of prostate cancer by biopsy. Biopsy tumor tissue from these patients was assayed.

All hypothesis tests were reported using two-sided p-values. To investigate if there is a significant relationship of outcomes (eg. clinical recurrence-free interval (cRFI), biochemical recurrence-free interval (bRFI), prostate cancer-specific survival (PCSS), overall survival (OS)) with individual genes, and demographic or clinical covariates), Cox Proportional Hazards (PH) models using maximum weighted pseudo partial-likelihood estimators were used and p-values from Wald tests of the null hypothesis that the hazard ratio (HR) is one are reported. To investigate if there is a significant relationship between individual genes and Gleason pattern of a particular sample, ordinal logistic regression models using maximum weighted pseudolikelihood methods were used and p-values from Wald tests of the null hypothesis that the odds ratio (OR) is one are reported. To investigate if there is a significant relationship between individual genes and upgrading and/or upstaging or adverse pathology at RP, logistic regression models using maximum weighted pseudolikelihood methods were used and p-values from Wald tests of the null hypothesis that the odds ratio (OR) is one are reported.

Coexpression Analysis

In an exemplary embodiment, the joint correlation of gene expression levels among prostate cancer specimens under study may be assessed. For this purpose, the correlation structures among genes and specimens may be examined through hierarchical cluster methods. This information may be used to confirm that genes that are known to be highly correlated in prostate cancer specimens cluster together as expected. Only genes exhibiting a nominally significant (unadjusted p<0.05) relationship with cRFI in the univariate Cox PH regression analysis are included in these analyses.

One skilled in the art will recognize that many co-expression analysis methods now known or later developed will fall within the scope and spirit of the present invention. These methods may incorporate, for example, correlation coefficients, co-expression network analysis, clique analysis, etc., and may be based on expression data from RT-PCR, microarrays, sequencing, and other similar technologies. For example, gene expression clusters can be identified using pair-wise analysis of correlation based on Pearson or Spearman correlation coefficients. (See, e.g., Pearson K. and Lee A., Biometrika 2, 357 (1902); C. Spearman, Amer. J. Psychol 15:72-101 (1904); J. Myers, A. Well, Research Design and Statistical Analysis, p. 508 (2nd Ed., 2003).) An exemplary method for identifying co-expressed genes is described in Example 3 below.

Normalization of Expression Levels

The expression data used in the methods disclosed herein can be normalized. Normalization refers to a process to correct for (normalize away), for example, differences in the amount of RNA assayed and variability in the quality of the RNA used, to remove unwanted sources of systematic variation in Ct or Cp measurements, and the like. With respect to RT-PCR experiments involving archived fixed paraffin embedded tissue samples, sources of systematic variation are known to include the degree of RNA degradation relative to the age of the patient sample and the type of fixative used to store the sample. Other sources of systematic variation are attributable to laboratory processing conditions.

Assays can provide for normalization by incorporating the expression of certain normalizing genes, which do not significantly differ in expression levels under the relevant conditions. Exemplary normalization genes disclosed herein include housekeeping genes. (See, e.g., E. Eisenberg, et al., Trends in Genetics 19(7):362-365 (2003).) Normalization can be based on the mean or median signal (Ct or Cp) of all of the assayed genes or a large subset thereof (global normalization approach). In general, the normalizing genes, also referred to as reference genes, are typically genes that are known not to exhibit meaningfully different expression in prostate cancer as compared to non-cancerous prostate tissue, and track with various sample and process conditions, thus provide for normalizing away extraneous effects.

In exemplary embodiments, one or more of the following genes are used as references by which the mRNA expression data is normalized: AAMP, ARF1, ATP5E, CLTC, GPS1, and PGK1. The calibrated weighted average CT or Cp measurements for each of the prognostic and predictive genes may be normalized relative to the mean of five or more reference genes.

Those skilled in the art will recognize that normalization may be achieved in numerous ways, and the techniques described above are intended only to be exemplary, not exhaustive.

Standardization of Expression Levels

The expression data used in the methods disclosed herein can be standardized. Standardization refers to a process to effectively put all the genes on a comparable scale. This is performed because some genes will exhibit more variation (a broader range of expression) than others. Standardization is performed by dividing each expression value by its standard deviation across all samples for that gene. Hazard ratios are then interpreted as the proportional change in the hazard for the clinical endpoint (clinical recurrence, biological recurrence, death due to prostate cancer, or death due to any cause) per 1 standard deviation increase in expression.

Kits of the Invention

The materials for use in the methods of the present invention are suited for preparation of kits produced in accordance with well-known procedures. The present disclosure thus provides kits comprising agents, which may include gene-specific or gene-selective probes and/or primers, for quantifying the expression of the disclosed genes for predicting prognostic outcome or response to treatment. Such kits may optionally contain reagents for the extraction of RNA from tumor samples, in particular fixed paraffin-embedded tissue samples and/or reagents for RNA amplification. In addition, the kits may optionally comprise the reagent(s) with an identifying description or label or instructions relating to their use in the methods of the present invention. The kits may comprise containers (including microliter plates suitable for use in an automated implementation of the method), each with one or more of the various materials or reagents (typically in concentrated form) utilized in the methods, including, for example, chromatographic columns, pre-fabricated microarrays, buffers, the appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP and dTTP; or rATP, rCTP, rGTP and UTP), reverse transcriptase, DNA polymerase, RNA polymerase, and one or more probes and primers of the present invention (e.g., appropriate length poly(T) or random primers linked to a promoter reactive with the RNA polymerase). Mathematical algorithms used to estimate or quantify prognostic or predictive information are also properly potential components of kits.

Reports

The methods of this invention, when practiced for commercial diagnostic purposes, generally produce a report or summary of information obtained from the herein-described methods. For example, a report may include information concerning expression levels of one or more genes, classification of the tumor or the patient's risk of recurrence, the patient's likely prognosis or risk classification, clinical and pathologic factors, and/or other information. The methods and reports of this invention can further include storing the report in a database. The method can create a record in a database for the subject and populate the record with data. The report may be a paper report, an auditory report, or an electronic record. The report may be displayed and/or stored on a computing device (e.g., handheld device, desktop computer, smart device, website, etc.). It is contemplated that the report is provided to a physician and/or the patient. The receiving of the report can further include establishing a network connection to a server computer that includes the data and report and requesting the data and report from the server computer.

Computer Program

The values from the assays described above, such as expression data, can be calculated and stored manually. Alternatively, the above-described steps can be completely or partially performed by a computer program product. The present invention thus provides a computer program product including a computer readable storage medium having a computer program stored on it. The program can, when read by a computer, execute relevant calculations based on values obtained from analysis of one or more biological samples from an individual (e.g., gene expression levels, normalization, standardization, thresholding, and conversion of values from assays to a score and/or text or graphical depiction of tumor stage and related information). The computer program product has stored therein a computer program for performing the calculation.

The present disclosure provides systems for executing the program described above, which system generally includes: a) a central computing environment; b) an input device, operatively connected to the computing environment, to receive patient data, wherein the patient data can include, for example, expression level or other value obtained from an assay using a biological sample from the patient, or microarray data, as described in detail above; c) an output device, connected to the computing environment, to provide information to a user (e.g., medical personnel); and d) an algorithm executed by the central computing environment (e.g., a processor), where the algorithm is executed based on the data received by the input device, and wherein the algorithm calculates an expression score, thresholding, or other functions described herein. The methods provided by the present invention may also be automated in whole or in part.

Having described the invention, the same will be more readily understood through reference to the following Examples, which are provided by way of illustration, and are not intended to limit the invention in any way.

EXAMPLES Example 1: Selection of 81 Genes for Algorithm Development

A gene identification study to identify genes associated with clinical recurrence, biochemical recurrence and/or death from prostate cancer is described in U.S. Provisional Application Nos. 61/368,217, filed Jul. 27, 2010; 61/414,310, filed Nov. 16, 2010; and 61/485,536, filed May 12, 2011, and in U.S. Pub. No. 20120028264, filed Jul. 25, 2011, and published Feb. 2, 2012 (all of which are hereby incorporated by reference). RT-PCR analysis was used to determine RNA expression levels for 732 genes and reference genes in prostate cancer tissue and surrounding normal appearing tissue (NAT) in patients with early-stage prostate cancer treated with radical prostatectomy. Genes significantly associated (p<0.05) with clinical recurrence-free interval (cRFI), biochemical recurrence-free interval (bRFI), prostate cancer-specific survival (PCSS), and upgrading/upstaging were determined.

From the genes that were identified as being associated with outcome, 81 genes were selected for subsequent algorithm development. The primers, probes, and amplicon sequences of the 81 genes (and 5 reference genes) are listed in Table A. The genes selected were among the most prognostic with respect to cRFI and other properties and shown in Tables 1A-1B. Other properties considered were: 1) Strongest genes with respect to the regression to the mean corrected standardized hazard ratio for the association of gene expression and cRFI in the primary Gleason pattern tumor; 2) Consistency in association (hazard ratio) with cRFI using the highest Gleason pattern tumor; 3) Associated with prostate-cancer specific survival (PCSS); 4) Strong hazard ratio after adjustment for The University of San Francisco Cancer of the Prostate Risk Assessment (CAPRA) (Cooperberg et al., J. Urol. 173:1983-1942, 2005); 5) Statistically significant odds ratio for the association between gene expression and surgical Gleason pattern of the tumor; 6) Large overall variability with greater between-patient variability than within-patient variability preferable; and 7) Highly expressed.

The true discovery rate degree of association (TDRDA) method (Crager, Stat Med. 2010 January 15; 29(1):33-45.) was used in the analysis of gene expression and cRFI and results are shown in Table 1A. The true discovery rate is the counterpart to the false discovery rate. Univariate Cox PH regression models were fit and the TDRDA method was used to correct estimated standardized hazard ratios for regression to the mean (RM) and and assess false discovery rates for identification of genes with absolute standardized hazard ratio of at least a specified level. The false discovery rates were controlled at 10%. The TDRDA method identifies sets of genes among which a specified proportion are expected to have an absolute association (here, the absolute standardized hazard ratio) of a specified degree or more. This leads to a gene ranking method that uses the maximum lower bound (MLB) degree of association for which each gene belongs to a TDRDA set. Estimates of each gene's actual degree of association with approximate correction for “selection bias” due to regression to the mean can be derived using simple bivariate normal theory and Efron and Tibshirani's empirical Bayes approach. Efron, Annals of Applied Statistics 2:197-223 (2008); Efron and Tibshirani. Genetic Epidemiology 23: 70-86. Table 1A shows the RM-corrected estimate of the standardized hazard ratio and the MLB for each gene using either the primary Gleason pattern (PGP) or highest Gleason pattern (HGP) sample gene expression. Genes marked with a direction of association of −1 are associated with a reduced likelihood of clinical recurrence, while those marked with a direction of association of 1 are associated with an increased likelihood of clinical recurrence.

Within patient and between patient variance components were estimated using a mixed model treating the patient effect as random. The overall mean and standard deviation of normalized gene expression as well as within- and between-patient components of variance are shown in Table 1A.

Univariate Cox PH regression models using maximum weighted partial pseudolikelihood estimation were used to estimate the association between gene expression and prostate cancer specific-survival (PCSS). The standardized hazard ratio (HR), p-value and q-value using Storey's FDR method are reported in Table 1B. Storey, Journal of the Royal Statistical Society, Series B 64:479-498 (2002). The q-value can be interpreted as the empirical Bayes posterior probability given the data that the gene identified is a false discovery, that is, the probability that it has no association with clinical recurrence.

Univariate ordinal logistic regression models were used to estimate the association between gene expression and the Gleason pattern of the primary Gleason pattern tumor (3, 4, 5). The standardized odds ratio (OR), p-value and q-value using Storey's FDR method are reported in Table 1B.

FIG. 1 shows an example of a dendrogram depicting the association of the 81 genes. The y-axis corresponds to the average distance between clusters measured as 1-Pearson r. The smaller the number (distance measure), the more highly correlated the genes. The amalgamation method is weighted pair-group average. Genes that were co-expressed were identified from the dendrogram and are grouped into gene groups. Based on FIG. 1, the genes from the Gene Identification study were formed into the following gene groups or subsets:

Cellular organization gene group (BIN1; IGF1; C7; GSN; DES; TGFB1I1; TPM2; VCL; FLNC; ITGA7; COL6A1; PPP1R12A; GSTM1; GSTM2; PAGE4; PPAP2B; SRD5A2; PRKCA; IGFBP6; GPM6B; OLFML3; HLF)

Basal epithelia gene group (CYP3A5; KRT15; KRT5; LAMB3; SDC1)

Stress response gene group (DUSP1; EGR1; FOS; JUN; EGR3; GADD45B; ZFP36)

Androgen gene group (FAM13C; KLK2; AZGP1; SRD5A2)

Stromal gene group (ASPN; SFRP4; BGN; THBS2; INHBA; COL1A1; COL3A1; COL1A2; SPARC; COL8A1; COL4A1; FN1; FAP; COL5A2)

Proliferation gene group (CDCl20; TPX2; UBE2T; MYBL2; CDKN2C)

TABLE 1A Association with Association with cR in PGP sample cR in HGP sample Direc- Direc- tion Absolute tion Mean SD Between- Within- of RM of normalized normalized patient patient Total Asso- Corrected Asso- Absolute RM GENE cp CP variance variance variance ciation HR MLB ciation Corrected HR MLB ARF1 11.656805 0.2475574 0.04488 0.01646 0.0613399 −1 1.0849132 — −1 1.2006385 1.0397705 ATP5E 10.896515 0.2667133 0.05141 0.01979 0.0711992 1 1.2599111 1.0908967 1 1.3877428 1.165325 CLTC 10.597008 0.1780634 0.01554 0.01619 0.0317257 1 1.0896793 1.002002 1 1.2292343 1.0554846 GPS1 9.2927019 0.2169116 0.03181 0.01528 0.0470897 1 1.0064191 — −1 1.1089053 — PGK1 8.3680642 0.2655009 0.04957 0.02099 0.0705517 1 1.1174152 1.0151131 1 1.2149168 1.0607752 ASPN 5.4846081 1.1701993 0.4981 0.8719 1.3699944 1 1.7716283 1.4276075 1 1.7114564 1.4007391 BGN 11.299746 0.7357491 0.3259 0.2159 0.5417276 1 1.6133066 1.3271052 1 1.7312777 1.4007391 COL1A1 11.325411 0.8840402 0.4748 0.3073 0.7821127 1 1.6162028 1.3284329 1 1.7982985 1.4304656 COL1A2 10.093055 0.8232027 0.4321 0.2461 0.6781941 1 1.1319748 1.0222438 1 1.3449362 1.136553 COL3A1 11.007109 0.7944239 0.352 0.2795 0.6315424 1 1.5695255 1.2969301 1 1.7133767 1.4007391 COL4A1 7.8408647 0.6731713 0.2393 0.2142 0.4534598 1 1.3297169 1.14225 1 1.4292283 1.1996142 COL5A2 5.2708574 0.9571692 0.4 0.5166 0.9166661 1 1.1715343 1.0408108 1 1.1822568 1.0408108 F2R 7.0775127 1.0110657 0.5529 0.47 1.022934 1 1.5019815 1.2636445 1 1.4888813 1.2361479 FAP 5.0493366 1.1898915 0.6577 0.759 1.4166546 1 1.3007869 1.1162781 1 1.3882726 1.1641602 FN1 9.5176438 0.7224014 0.3059 0.2163 0.5222401 1 1.0505668 — 1 1.1524617 1.0253151 INHBA 5.8059993 1.2653019 0.9629 0.6392 1.6021763 1 1.896185 1.4858693 1 2.1859455 1.7177237 SFRP4 7.8225007 1.2053184 0.7997 0.6541 1.4537934 1 1.5382115 1.2763443 1 1.5692525 1.2969301 SPARC 10.544556 0.7978856 0.4311 0.206 0.6371517 1 1.3683299 1.1711662 1 1.6187451 1.3324242 THBS2 4.7779897 1.0825934 0.7121 0.4608 1.1728864 1 1.5523887 1.2904616 1 1.6829249 1.3785056 BIN1 7.8741434 0.840604 0.4445 0.2627 0.7071728 −1 1.5631385 1.2930451 −1 1.3294226 1.1185129 C7 8.4895479 1.1083704 0.6917 0.5377 1.2293358 −1 1.5658393 1.2687092 −1 1.4724885 1.220182 COL6A1 7.3615421 0.848837 0.3381 0.3828 0.7209474 −1 1.5439152 1.2687092 −1 1.2634411 1.0746553 DES 11.967287 0.896286 0.4101 0.3938 0.8038418 −1 1.5007183 1.2386227 −1 1.3032825 1.0963648 FLNC 8.6795128 1.0679528 0.572 0.5692 1.1412391 −1 1.2696693 1.0650268 −1 1.2353942 1.0491707 GPM6B 7.9402089 0.9453416 0.4441 0.4501 0.8942266 −1 1.4471085 1.2056273 −1 1.4931412 1.2386227 GSN 8.8308175 0.7789199 0.3756 0.2316 0.6071864 −1 1.6223835 1.3073471 −1 1.3639212 1.136553 GSTM1 6.4175398 1.2720365 1.0519 0.5675 1.619374 −1 1.5226009 1.2560853 −1 1.5822193 1.2969301 GSTM2 7.2950478 1.0014875 0.5069 0.4967 1.0036007 −1 1.6694102 1.3284329 −1 1.4815895 1.220182 HLF 5.0774106 0.9618562 0.4516 0.4741 0.9257242 −1 1.6225351 1.2956338 −1 1.5817614 1.2891718 IGF1 7.6180418 1.1441945 0.7925 0.5177 1.3101729 −1 1.4732764 1.2165269 −1 1.6861196 1.359341 IGFBP6 7.0089783 1.0816262 0.6302 0.5405 1.1707038 −1 1.5534588 1.257342 −1 1.1860594 1.0273678 ITGA7 7.3299653 0.8913845 0.4034 0.3916 0.7950725 −1 1.5556326 1.2636445 −1 1.3587711 1.1308844 OLFML3 8.1932023 0.8189012 0.4 0.2711 0.6710997 −1 1.5254982 1.2649088 −1 1.3364894 1.1263699 PAGE4 7.406255 1.4889881 1.3023 0.9164 2.2187195 −1 1.6316984 1.2969301 −1 1.5178657 1.2435871 PPAP2B 8.8879191 0.7884647 0.3841 0.238 0.6221573 −1 1.5664582 1.2649088 −1 1.4703629 1.2068335 PPP1R12A 9.369152 0.5056735 0.1361 0.1198 0.2558759 −1 1.4273047 1.1711662 −1 1.3719705 1.1297541 PRKCA 7.4654299 0.7936779 0.2984 0.3319 0.6302916 −1 1.4498244 1.1960207 −1 1.2221961 1.0554846 SRD5A2 5.7878904 1.2691925 0.8776 0.7343 1.6119317 −1 1.8236528 1.4276075 −1 1.723879 1.4007391 VCL 8.9766979 0.720267 0.2667 0.2524 0.5191126 −1 1.526093 1.2423441 −1 1.4080433 1.1525766 TGFB1I1 8.2191469 0.7816114 0.3143 0.297 0.6113098 −1 1.4793989 1.2104595 −1 1.3608249 1.1229959 TPM2 11.83198 0.8626062 0.4118 0.3328 0.7446062 −1 1.5739312 1.2687092 −1 1.4709656 1.2116705 TPX2 4.552336 1.0856094 0.4888 0.6903 1.1791549 1 1.6454619 1.3444702 1 1.886703 1.4829005 CDC20 3.5608774 0.7971167 0.1978 0.4378 0.6356438 1 1.431589 1.232445 1 1.568564 1.3271052 CDKN2C 3.5214932 0.6898842 0.137 0.3391 0.476113 1 1.4751885 1.257342 1 1.6210275 1.3485096 MYBL2 3.7669784 0.981064 0.4061 0.5569 0.9629897 1 1.5421274 1.2969301 1 1.5553089 1.3086551 UBE2T 3.5015369 0.7220453 0.1927 0.3289 0.5215967 1 1.5156185 1.272521 1 1.2186754 1.0618365 CYP3A5 4.5549862 1.3365744 0.5786 1.2085 1.7871462 −1 1.851997 1.4276075 −1 1.8669598 1.4304656 KRT15 8.1889409 1.8188542 1.3539 1.956 3.3098961 −1 1.4985779 1.2423441 −1 1.7380356 1.4007391 KRT5 7.046586 1.528426 0.8388 1.4983 2.3371203 −1 1.4656121 1.2140963 −1 1.6810573 1.3702593 LAMB3 6.3566958 1.3451305 0.6358 1.1744 1.8101739 −1 1.435434 1.1996142 −1 1.5003356 1.2287532 EGR1 12.925851 1.0521413 0.7737 0.3343 1.1079529 −1 1.5840317 1.2687092 −1 1.5773643 1.2801791 FOS 12.383619 1.1226481 0.8869 0.3746 1.2614295 −1 1.580311 1.2687092 −1 1.655218 1.3310925 GADD45B 8.8760029 1.2001535 0.893 0.5485 1.4414667 −1 1.5331503 1.2460767 −1 1.490302 1.220182 JUN 11.184249 1.027684 0.8099 0.2473 1.0571304 −1 1.5816073 1.2649088 −1 1.5652799 1.2649088 ZFP36 12.472841 1.1025865 0.8095 0.4072 1.2167102 −1 1.5169178 1.2423441 −1 1.580749 1.2840254 DUSP1 11.51936 0.8297195 0.439 0.25 0.6889744 −1 1.3986201 1.1502738 −1 1.40278 1.1525766 EGR3 9.7545461 1.3366461 1.2104 0.5778 1.7881414 −1 1.5114362 1.2349124 −1 1.4927024 1.2226248 FAM13C 7.4923611 0.9318455 0.58 0.2891 0.8690619 −1 1.678403 1.3716303 −1 1.7981441 1.4520843 KLK2 14.718412 0.6677811 0.3087 0.1376 0.4463114 −1 1.4443314 1.1936311 −1 1.4966995 1.2361479 ALDH1A2 5.8436909 1.0515294 0.4984 0.6079 1.1063379 −1 1.5394232 1.2649088 −1 1.817606 1.4304656 AZGP1 9.3508493 1.4900096 1.6597 0.5625 2.2222058 −1 1.6202418 1.329762 −1 1.4723607 1.2398619 ANPEP 7.1080338 2.3433136 3.865 1.6309 5.4959682 −1 1.5469141 1.257342 −1 1.7787926 1.4035433 AR 8.3681098 0.5094853 0.1629 0.09684 0.2597757 −1 1.0685968 — −1 1.1146402 1.004008 BMP6 4.7641658 1.1079887 0.5639 0.6645 1.2283377 1 1.5010706 1.2649088 1 1.7140049 1.4007391 CD276 8.9879201 0.5574321 0.168 0.1429 0.3109408 1 1.459777 1.2398619 1 1.6372299 1.3525612 CD44 7.5506865 1.1157404 0.8611 0.3849 1.2459543 −1 1.6840393 1.3444702 −1 1.5715077 1.3008267 COL8A1 7.1807327 0.9924731 0.529 0.4566 0.9856651 1 1.4782028 1.251071 1 1.6302371 1.3337573 CSF1 5.604685 0.806399 0.2828 0.3678 0.6506271 −1 1.4727627 1.2092496 −1 1.3423853 1.1162781 SRC 7.4585136 0.6681318 0.2723 0.1745 0.446735 −1 1.4782469 1.2435871 −1 1.5104712 1.2687092 CSRP1 12.967068 0.8434745 0.267 0.4448 0.711785 −1 1.2323596 1.0607752 −1 1.2436215 1.0639623 DPP4 6.5096496 1.228945 0.9313 0.5802 1.5114514 −1 1.5818046 1.2930451 −1 1.539425 1.2788996 TNFRSF10B 5.8731042 0.836448 0.3338 0.3663 0.7000558 −1 1.5759506 1.2687092 −1 1.4449471 1.2032184 ERG 7.2194906 2.2910776 4.1737 1.0805 5.2541704 1 1.0907212 1.0030045 1 1.0828357 — FAM107A 4.9863267 1.128037 0.6041 0.6691 1.2732255 −1 1.7326232 1.3539145 −1 1.5773305 1.2801791 IGFBP2 9.855496 0.7862553 0.4734 0.1454 0.6187898 −1 1.6368294 1.2982277 −1 1.4976178 1.2287532 CADM1 7.5914375 0.7603875 0.3308 0.2478 0.5785959 −1 1.6923754 1.3539145 −1 1.7428282 1.4035433 IL6ST 10.355178 0.5509714 0.1851 0.1187 0.3038017 −1 1.5709644 1.2687092 −1 1.470333 1.217744 LGALS3 8.5926121 0.7862207 0.3442 0.2743 0.6185665 −1 1.5048215 1.2411024 −1 1.3574436 1.1320159 SMAD4 8.902039 0.4085988 0.1025 0.06456 0.1670791 −1 1.669624 1.329762 −1 1.6100555 1.3112751 NFAT5 9.2271297 0.5003181 0.1647 0.08587 0.2505217 −1 1.7299501 1.3539145 −1 1.5858292 1.2995265 SDC1 7.2405046 0.9486094 0.4431 0.4573 0.9004048 1 1.1878437 1.0502204 1 1.0110389 — SHMT2 7.3818144 0.5716985 0.1376 0.1894 0.3270085 1 1.5185392 1.2687092 1 1.494364 1.2448313 SLC22A3 8.8366285 1.3865065 1.3128 0.6112 1.9240432 −1 1.6100215 1.2930451 −1 1.6531341 1.3271052 STAT5B 7.443638 0.479107 0.09118 0.1385 0.2296576 −1 1.4932136 1.2435871 −1 1.4376605 1.1948253 MMP11 4.0974635 1.1790067 0.6512 0.7396 1.3908598 1 1.4849754 1.257342 1 1.3586058 1.1514246 TUBB2A 8.3247821 0.9300317 0.553 0.3126 0.8656511 −1 1.4310473 1.1699956 −1 1.4520775 1.1817543

TABLE 1B Association with Association with Association with PCSS Endpoint cRFI, CAPRA Adjusted primary Gleason pattern Wald p- Storey q- Wald p- Storey q- Wald p- Storey q- GENE Std. HR value value Std. HR value value Std. OR value value ARF1 0.9976193 0.9892903 0.5842039 1.13407667 0.83255536 0.4298654 0.982273 0.907845 0.3887901 ATP5E 1.7881176 0.0115817 0.0404889 2.78746082 0.07918786 0.0876892 1.3546007 0.0347604 0.0377781 CLTC 1.026701 0.8673167 0.5533326 1.95450035 0.43611016 0.2800117 1.293925 0.4450274 0.2418238 GPS1 0.849428 0.3590422 0.3461617 1.04907052 0.93720193 0.4574281 0.8271353 0.1863038 0.1358186 PGK1 0.9801478 0.9057919 0.5657924 2.40228939 0.08542284 0.0920345 0.9775945 0.8943649 0.3866666 ASPN 3.0547166 1.85E−08 3.97E−06 1.98680704 3.75E−07 8.71E−06 2.6178187 3.32E−07 2.89E−06 BGN 2.6395972 2.34E−06 0.000144 2.65751131 3.29E−07 8.71E−06 2.5773715 1.19E−07 1.34E−06 COL1A1 2.5740243 1.16E−07 0.0000124 2.43783157 3.46E−09 3.73E−07 2.2304742 2.40E−06 0.0000138 COL1A2 1.6067045 0.0108778 0.0393063 1.30239373 0.14922155 0.1339808 1.1226733 0.4115421 0.2324622 COL3A1 2.3815758 7.22E−06 0.0002217 2.52337548 6.28E−08 3.29E−06 1.9034844 0.0001218 0.0003527 COL4A1 1.9704368 0.0008215 0.006465 2.14028481 0.00036078 0.0014267 1.0893045 0.5143164 0.2654536 COL5A2 1.9382474 0.0018079 0.0111059 1.20272948 0.23650362 0.1864588 1.1234816 0.4230095 0.2350912 F2R 2.1687429 0.000107 0.0016437 1.62196106 0.00161154 0.0046735 2.3718651 0.00000433 0.0000227 FAP 1.9932031 0.0015781 0.0098344 1.37558042 0.00581828 0.0124219 2.3624961 2.06E−07 2.16E−06 FN1 1.5366589 0.0242031 0.0667138 1.46470831 0.06004749 0.0723063 0.9406955 0.6630807 0.3149629 INHBA 3.0596839 1.07E−07 0.0000124 1.98607554 4.28E−09 3.73E−07 2.5487503 5.65E−06 0.0000273 SFRP4 2.3836087 0.0000248 0.0005927 1.67750397 1.87E−05 0.0001515 2.6895594 6.50E−11 1.80E−09 SPARC 2.249132 0.0000652 0.0012192 1.81223384 0.00166372 0.0047457 1.4031045 0.0236369 0.0279596 THBS2 2.5760475 2.97E−07 0.0000256 1.89140939 7.54E−07 0.0000146 1.8704603 0.0000261 0.0000967 BIN1 0.6582912 0.0008269 0.006465 0.53215394 1.35E−07 4.02E−06 0.4346961 2.11E−09 4.27E−08 C7 0.5305767 4.96E−06 0.0001778 0.61660218 1.43E−05 0.0001297 0.3687542 3.26E−12 1.24E−10 COL6A1 0.6814495 0.0146421 0.0470118 0.59821729 8.17E−05 0.0004585 0.4431674 2.65E−07 2.52E−06 DES 0.730098 0.0532354 0.1095273 0.61335977 0.00103514 0.0033666 0.3442483 2.78E−08 3.52E−07 FLNC 0.741509 0.0356714 0.0847442 0.85002893 0.23258502 0.1843726 0.3263553 4.74E−09 8.68E−08 GPM6B 0.6663566 0.0048803 0.0233566 0.60320026 0.00006446 0.0003935 0.4814972 2.01E−06 0.0000117 GSN 0.6464018 0.0057152 0.0257736 0.45512167 1.29E−07 4.02E−06 0.463566 3.08E−07 2.84E−06 GSTM1 0.6720834 0.0063399 0.0269917 0.61970734 4.44E−06 0.000058 0.4449932 1.22E−09 2.65E−08 GSTM2 0.514483 0.0000907 0.0015002 0.52721579 4.50E−06 0.000058 0.2939022 3.94E−13 2.00E−11 HLF 0.5812615 0.0004971 0.0047504 0.52096012 2.14E−06 0.0000338 0.4179279 8.18E−09 1.31E−07 IGF1 0.6118674 0.0001721 0.0022429 0.62158322 7.65E−06 0.0000807 0.3470943 2.24E−13 1.36E−11 IGFBP6 0.5776972 0.003161 0.0172052 0.60163498 7.96E−05 0.0004543 0.4536368 1.44E−08 2.18E−07 ITGA7 0.6760378 0.0331669 0.0810327 0.54167205 2.10E−05 0.0001661 0.3682462 4.07E−10 1.03E−08 OLFML3 0.6460637 0.0011279 0.0080836 0.57873289 0.00001453 0.0001297 0.4154584 1.88E−08 2.64E−07 PAGE4 0.5182669 5.75E−06 0.0001903 0.66287751 2.46E−06 0.0000357 0.2677212 2.80E−17 8.51E−15 PPAP2B 0.5680087 0.0006371 0.0055913 0.45475585 6.81E−06 0.0000765 0.4140322 4.85E−09 8.68E−08 PPP1R12A 0.6937407 0.0165382 0.0496415 0.46377868 0.00149833 0.0044262 0.4793933 0.0000198 0.0000772 PRKCA 0.6323113 0.0014455 0.0092768 0.52602482 6.68E−05 0.0004008 0.3682007 6.36E−09 1.07E−07 SRD5A2 0.4878954 2.93E−06 0.0001573 0.53197502 1.48E−09 2.58E−07 0.2848852 1.63E−14 1.45E−12 VCL 0.6936282 0.4610739 0.3983524 0.48920691 0.00010426 0.0005415 0.4393103 7.90E−06 0.0000369 TGEB1I1 0.6700491 0.0291804 0.0749233 0.58638267 0.00360838 0.0089058 0.3711508 1.96E−08 2.64E−07 TPM2 0.6225776 0.0050533 0.0236188 0.55889674 0.00020286 0.0008936 0.3008674 1.04E−09 2.44E−08 TPX2 2.07392 0.0416277 0.0942101 1.80670715 7.56E−08 3.29E−06 2.1153062 4.98E−06 0.0000248 CDC20 1.7300441 0.0000725 0.0012988 1.94643748 4.81E−06 0.0000598 1.6858143 0.000045 0.0001519 CDKN2C 1.99305 0.0125796 0.0432737 2.2133602 1.45E−05 0.0001297 1.6207388 0.00039 0.0009879 MYBL2 1.7372773 0.012916 0.0440786 1.64874559 6.39E−06 0.0000745 1.4091306 0.0068668 0.0098934 UBE2T 1.898363 0.0847937 0.1462198 1.87982565 0.00012433 0.0005929 1.594593 0.0006065 0.0014404 CYP3A5 0.5067698 0.0003008 0.0032333 0.56312246 1.39E−07 4.02E−06 0.5204925 9.40E−06 0.0000426 KRT15 0.6841526 0.0052343 0.0242015 0.7687095 7.50E−05 0.000435 0.5603937 0.0000173 0.0000713 KRT5 0.6729884 0.0041853 0.0216827 0.7264908 0.0002245 0.0009645 0.6401711 0.000719 0.0016558 LAMB3 0.7403354 0.0266386 0.0724973 0.76722056 0.00366663 0.0089858 0.730357 0.0177696 0.0219488 EGR1 0.4902253 0.0003344 0.0034238 0.60204599 1.58E−05 0.0001377 0.5936819 0.0007056 0.0016501 FOS 0.5555161 0.0045741 0.0223508 0.6374611 5.34E−05 0.0003316 0.5788589 0.0002156 0.0005851 GADD45B 0.5541679 0.0021788 0.0124919 0.64836478 3.81E−05 0.0002707 0.6176176 0.002403 0.0043482 JUN 0.505934 0.0013437 0.0088891 0.54410296 1.78E−05 0.00015 0.4795161 3.99E−07 3.37E−06 ZFP36 0.5757824 0.001207 0.0083714 0.66520845 0.00023482 0.0009966 0.6470667 0.0043499 0.0068874 DUSP1 0.6603212 0.0498518 0.1055975 0.63670824 0.00347613 0.0086407 0.586205 0.0007564 0.0017289 EGR3 0.5613678 0.0009351 0.0071803 0.72134117 0.0008831 0.002955 0.7186042 0.0260364 0.0300953 FAM13C 0.5260925 4.01E−09 1.72E−06 0.52541845 6.04E−10 2.10E−07 0.3709836 5.73E−11 1.74E−09 KLK2 0.5808923 0.0001686 0.0022429 0.56994638 0.00194411 0.0053697 0.5968229 0.0002289 0.0006158 ALDH1A2 0.5608861 0.0000177 0.0004751 0.65303146 0.00010806 0.000545 0.2822456 4.34E−07 3.57E−06 AZGP1 0.6167537 3.96E−06 0.0001778 0.6708779 4.29E−07 9.33E−06 0.5150783 1.17E−06 7.43E−06 ANPEP 0.5313085 0.0008229 0.006465 0.80509148 0.00011244 0.000559 0.6761885 0.0081251 0.0114354 AR 0.9479643 0.7435374 0.510526 0.77328075 0.35466409 0.244458 0.9337631 0.6073904 0.295812 BMP6 1.4900185 0.0210638 0.060383 1.44999312 0.00201534 0.0054792 2.3713254 6.85E−07 4.86E−06 CD276 1.6684232 0.0049028 0.0233566 2.16998768 0.00057532 0.0020641 2.1955258 2.75E−06 0.0000155 CD44 0.6866428 0.0155666 0.0485038 0.5502693 1.06E−07 4.02E−06 0.7793905 0.0682278 0.0654298 COL8A1 2.2449967 0.0000271 0.0006141 1.91011656 4.90E−05 0.0003216 1.8755907 9.13E−06 0.0000421 CSF1 0.6749873 0.0193771 0.0562984 0.44321491 5.01E−07 0.0000103 0.9573718 0.7643438 0.3461448 SRC 0.6670294 0.0040025 0.0212478 0.47320013 1.67E−06 0.0000307 0.766355 0.0813478 0.0758976 CSRP1 0.7112339 0.0067019 0.0277098 0.89239013 0.2633928 0.1994666 0.4248705 0.0058729 0.0088825 DPP4 0.5441442 1.12E−06 0.00008 0.68915455 0.00013991 0.0006492 0.4140282 1.92E−06 0.0000114 TNFRSF10B 0.6852925 0.0143692 0.0468086 0.53054603 4.04E−05 0.0002757 0.7430912 0.0304132 0.0339912 ERG 1.0765349 0.6794217 0.4926667 1.12737455 0.0349341 0.0496809 0.8943961 0.4148417 0.2324622 FAM107A 0.540565 0.0000605 0.0011827 0.57090059 6.82E−08 3.29E−06 0.3476335 1.99E−08 2.64E−07 IGFBP2 0.6977969 0.0532257 0.1095273 0.45025927 6.42E−06 0.0000745 0.6063083 0.0001586 0.0004465 CADM1 0.6456383 0.0150546 0.0472518 0.40819615 3.14E−08 2.19E−06 0.5598139 0.0001184 0.000346 IL6ST 0.5740052 0.0003647 0.0036466 0.33440325 2.36E−06 0.0000357 0.5462964 0.0040541 0.0065556 LGALS3 0.6782394 0.0071303 0.0283525 0.53406803 5.18E−05 0.0003278 0.5903729 0.0030449 0.0052894 SMAD4 0.5277628 4.87E−06 0.0001778 0.24376793 2.03E−06 0.0000336 0.3346823 1.85E−06 0.0000112 NFAT5 0.5361732 0.0000856 0.0014722 0.20926313 3.51E−07 8.71E−06 0.5518236 0.0000356 0.000126 SDC1 1.7097015 0.007187 0.0283525 1.43080815 0.02325931 0.0359744 1.6597668 0.0010445 0.002268 SHMT2 1.9491131 0.0031065 0.0171257 1.94514573 0.00591315 0.0124714 1.6896076 0.0074605 0.0105488 SLC22A3 0.5168636 0.000117 0.001706 0.65464678 7.32E−06 0.0000796 0.2293355 1.91E−14 1.45E−12 STAT5B 0.7002104 0.0396042 0.0914258 0.44673718 0.00045662 0.0017462 0.5417213 0.0000465 0.0001553 MMP11 1.8691119 0.0001041 0.0016437 1.62300343 1.23E−05 0.0001222 2.3250222 7.87E−07 5.44E−06 TUBB2A 0.6134538 0.0026235 0.0148438 0.56476388 1.81E−05 0.00015 0.9566513 0.7630842 0.3461448

Example 2: Algorithm Development Based on Data from A Companion Study

The Cleveland Clinic (“CC”) Companion study consists of three patient cohorts and separate analyses for each cohort as described in Table 2. The first cohort (Table 2) includes men with low to high risk (based on AUA criteria) prostate cancer from Gene ID study 09-002 who underwent RP at CC between 1987 and 2004 and had diagnostic biopsy tissue available at CC. Cohorts 2 and 3 include men with clinically localized Low and Intermediate Risk (based on AUA criteria) prostate cancer, respectively, who might have been reasonable candidates for active surveillance but who underwent radical prostatectomy (RP) within 6 months of the diagnosis of prostate cancer by biopsy. The main objective of Cohort 1 was to compare the molecular profile from biopsy tissue with that from radical prostatectomy tissue. The main objective of Cohorts 2 and 3 was to develop a multigene predictor of upgrading/upstaging at RP using biopsy tissue in low to intermediate risk patients at diagnosis.

Matched biopsy samples were obtained for a subset of the patients (70 patients) from the gene identification study. Gene expression of the 81 selected genes and the 5 reference genes (ARF1, ATP5E, CLTC, GPS1, PGK1) were compared in the RP specimens and the biopsy tissue obtained from these 70 patients.

The 81 genes were evaluated in Cohorts 2 and 3 for association with upgrading and upstaging. The association between these 81 genes and upgrading and upstaging in Cohorts 2 and 3 are shown in Table 3. P values and standardized odds ratio are provided.

In this context, “upgrade” refers to an increase in Gleason grade from 3+3 or 3+4 at the time of biopsy to greater than or equal to 3+4 at the time of RP. “Upgrade2” refers to an increase in Gleason grade from 3+3 or 3+4 at the time of biopsy to greater than or equal to 4+3 at the time of RP.

TABLE 2 Cohort # Cohort Description # of Patients Objectives 1 Subset of patients from Gene ID study 70 Comparison of gene expression from 09-002 who underwent RP at CC between biopsy sample with gene expression 1987 and 2004 and had diagnostic biopsy from RP specimen (Co-Primary tissue available at CC. Objective) Patients from the original stratified cohort Explore association of risk of sample with available biopsy tissue blocks recurrence after RP with gene expression from biopsy sample and gene expression from RP sample Explore association of risk of recurrence after RP with gene expression from RP samples 2 Low Risk Patients from CC database of 92 Association between gene expression patients who were biopsied, and then from biopsy sample and likelihood of underwent RP at CC between 1999 and upgrading/upstaging in tissue obtained 2010 at prostatectomy All patients in database who meet (Co-Primary Objective) minimum tumor tissue criteria 3 Intermediate Risk Patients from CC 75 Association between gene expression database of patients who were biopsied, from biopsy sample and likelihood of and then underwent RP at CC between upgrading/upstaging in tissue obtained 1999 and 2010 at prostatectomy All patients in database who meet minimum tumor tissue criteria

Several different models were explored to compare expression between the RP and biopsy specimens. Genes were chosen based on consistency of expression between the RP and biopsy specimens. FIGS. 2A-2E are the scatter plots showing the comparison of normalized gene expression (Cp) for matched samples from each patient where the x-axis is the normalized gene expression from the PGP RP sample (PGP) and the y-axis is the normalized gene expression from the biopsy sample (BX). FIGS. 3A-3D show range plots of gene expression of individual genes within each gene group in the biopsy (BX) and PGP RP samples.

After evaluating the concordance of gene expression in biopsy and RP samples, the following algorithms (RS models) shown in Table 4 were developed where the weights are determined using non-standardized, but normalized data. Some genes, such as SRD5A2 and GSTM2, which fall within the cellular organization gene group, were also evaluated separately and independent coefficients were assigned (see the “other” category in Table 4). In other instances, GSTM1 and GSMT2 were grouped as an oxidative “stress” group and a coefficient was assigned to this “stress” group (see RS20 and RS22 models). Other genes, such as AZGP1 and SLC22A3, which did not fall within any of the gene groups, were also included in certain algorithms (see the “other” category in Table 4). Furthermore, the androgen gene group was established to include FAM13C, KLK2, AZGP1, and SRD5A2. Some genes such as BGN, SPARC, FLNC, GSN, TPX2 and SRD5A2 were thresholded before being evaluated in models. For example, normalized expression values below 4.5 were set to 4.5 for TPX2 and normalized expression values below 5.5 were set to 5.5 for SRD5A2.

TABLE 3 Association between the 81 genes and Upgrading and Upstaging in Cohorts 2/3 p-value Std OR 95% CI p-value Std OR 95% CI p-value Std OR 95% CI Gene N UpGrade Upgrade Upgrade Upgrade2 Upgrade2 Upgrade2 Upstage Upstage Upstage ALDH1A2 167 0.501 1.11 (0.82, 1.52) 0.932 1.02 (0.70, 1.47) 0.388 0.86 (0.61, 1.22) ANPEP 167 0.054 1.36 (0.99, 1.87) 0.933 0.98 (0.68, 1.42) 0.003 0.58 (0.40, 0.83) AR 167 0.136 1.27 (0.93, 1.74) 0.245 0.81 (0.56, 1.16) 0.005 0.60 (0.42, 0.86) ARF1 167 0.914 0.98 (0.72, 1.34) 0.051 1.45 (1.00, 2.11) 0.371 1.17 (0.83, 1.66) ASPN 167 0.382 1.15 (0.84, 1.56) 0.040 1.60 (1.02, 2.51) 0.069 1.46 (0.97, 2.19) ATP5E 167 0.106 1.30 (0.95, 1.77) 0.499 0.88 (0.61, 1.27) 0.572 0.90 (0.64, 1.28) AZGP1 167 0.192 1.23 (0.90, 1.68) 0.190 0.79 (0.55, 1.13) 0.005 0.59 (0.41, 0.85) BGN 167 0.568 0.91 (0.67, 1.25) 0.001 2.15 (1.39, 3.33) 0.020 1.56 (1.07, 2.28) BIN1 167 0.568 1.09 (0.80, 1.49) 0.634 0.92 (0.64, 1.32) 0.104 0.75 (0.54, 1.06) BMP6 167 0.509 0.90 (0.66, 1.23) 0.015 1.59 (1.09, 2.30) 0.650 1.08 (0.77, 1.54) C7 167 0.677 1.07 (0.78, 1.46) 0.013 1.66 (1.11, 2.47) 0.223 0.80 (0.56, 1.14) CADM1 167 0.082 0.74 (0.52, 1.04) 0.235 0.81 (0.57, 1.15) 0.039 0.69 (0.48, 0.98) CD276 167 0.454 0.89 (0.65, 1.21) 0.362 0.84 (0.58, 1.22) 0.214 1.25 (0.88, 1.78) CD44 167 0.122 1.28 (0.94, 1.75) 0.305 1.23 (0.83, 1.81) 0.876 0.97 (0.69, 1.38) CDC20 166 0.567 1.10 (0.80, 1.50) 0.298 1.21 (0.84, 1.75) 0.279 1.21 (0.86, 1.71) CDKN2C 152 0.494 0.89 (0.64, 1.24) 0.908 0.98 (0.67, 1.43) 0.834 1.04 (0.72, 1.49) CLTC 167 0.102 0.76 (0.55, 1.06) 0.300 0.82 (0.57, 1.19) 0.264 0.82 (0.58, 1.16) COL1A1 167 0.732 1.06 (0.77, 1.44) 0.000 3.04 (1.93, 4.79) 0.006 1.65 (1.15, 2.36) COL1A2 167 0.574 0.91 (0.67, 1.25) 0.017 1.65 (1.09, 2.50) 0.521 0.89 (0.63, 1.26) COL3A1 167 0.719 0.94 (0.69, 1.29) 0.000 2.98 (1.88, 4.71) 0.020 1.53 (1.07, 2.20) COL4A1 167 0.682 0.94 (0.69, 1.28) 0.000 2.12 (1.39, 3.22) 0.762 0.95 (0.67, 1.35) COL5A2 167 0.499 1.11 (0.82, 1.52) 0.009 1.81 (1.16, 2.83) 0.516 0.89 (0.63, 1.26) COL6A1 167 0.878 0.98 (0.72, 1.33) 0.001 2.14 (1.37, 3.34) 0.883 1.03 (0.72, 1.46) COL8A1 165 0.415 0.88 (0.64, 1.20) 0.000 3.24 (1.88, 5.61) 0.044 1.51 (1.01, 2.25) CSF1 167 0.879 1.02 (0.75, 1.40) 0.187 1.31 (0.88, 1.96) 0.110 0.76 (0.54, 1.07) CSRP1 165 0.258 1.20 (0.87, 1.65) 0.226 1.26 (0.87, 1.82) 0.641 0.92 (0.65, 1.31) CYP3A5 167 0.989 1.00 (0.73, 1.36) 0.188 1.28 (0.88, 1.87) 0.937 1.01 (0.71, 1.44) DES 167 0.776 1.05 (0.77, 1.43) 0.088 1.40 (0.95, 2.05) 0.242 0.81 (0.57, 1.15) DPP4 167 0.479 0.89 (0.65, 1.22) 0.005 0.60 (0.42, 0.85) 0.000 0.51 (0.36, 0.74) DUSP1 167 0.295 0.84 (0.61, 1.16) 0.262 0.82 (0.58, 1.16) 0.427 0.87 (0.62, 1.22) EGR1 167 0.685 0.94 (0.69, 1.28) 0.217 1.27 (0.87, 1.85) 0.370 1.18 (0.83, 1.68) EGR3 166 0.025 0.69 (0.50, 0.95) 0.539 0.89 (0.62, 1.29) 0.735 1.06 (0.75, 1.51) ERG 166 0.002 0.58 (0.42, 0.81) 0.000 0.42 (0.28, 0.64) 0.768 1.05 (0.74, 1.50) F2R 160 0.324 0.85 (0.62, 1.17) 0.009 1.77 (1.16, 2.70) 0.000 2.39 (1.52, 3.76) FAM107A 143 0.832 1.04 (0.74, 1.45) 0.088 1.42 (0.95, 2.11) 0.687 1.08 (0.74, 1.58) FAM13C 167 0.546 1.10 (0.81, 1.50) 0.041 0.68 (0.47, 0.98) 0.003 0.58 (0.40, 0.83) FAP 167 0.540 0.91 (0.67, 1.24) 0.093 1.37 (0.95, 1.97) 0.001 1.85 (1.28, 2.68) FLNC 167 0.963 1.01 (0.74, 1.37) 0.254 1.26 (0.85, 1.87) 0.030 0.68 (0.48, 0.96) FN1 167 0.530 0.91 (0.66, 1.23) 0.005 1.73 (1.18, 2.53) 0.364 1.17 (0.83, 1.66) FOS 167 0.649 0.93 (0.68, 1.27) 0.071 1.38 (0.97, 1.97) 0.015 1.53 (1.09, 2.16) GADD45B 167 0.978 1.00 (0.73, 1.36) 0.105 1.38 (0.94, 2.04) 0.876 0.97 (0.69, 1.38) GPM6B 159 0.944 0.99 (0.72, 1.36) 0.002 1.95 (1.27, 2.97) 0.266 0.81 (0.57, 1.17) GPS1 167 0.404 1.14 (0.84, 1.56) 0.609 0.91 (0.62, 1.32) 0.125 1.31 (0.93, 1.86) GSN 167 0.272 0.84 (0.61, 1.15) 0.309 0.83 (0.57, 1.19) 0.027 0.67 (0.47, 0.96) GSTM1 167 0.178 1.24 (0.91, 1.69) 0.762 0.95 (0.66, 1.36) 0.000 0.50 (0.34, 0.72) GSTM2 167 0.145 1.26 (0.92, 1.73) 0.053 1.48 (1.00, 2.20) 0.654 0.92 (0.65, 1.31) HLF 167 0.979 1.00 (0.73, 1.36) 0.602 1.11 (0.76, 1.62) 0.030 0.69 (0.49, 0.96) IGF1 167 0.313 1.17 (0.86, 1.60) 0.878 0.97 (0.67, 1.40) 0.146 0.77 (0.55, 1.09) IGFBP2 167 0.253 1.20 (0.88, 1.64) 0.493 0.88 (0.61, 1.27) 0.051 0.70 (0.49, 1.00) IGFBP6 167 0.336 0.86 (0.62, 1.17) 0.510 1.14 (0.78, 1.66) 0.204 0.80 (0.57, 1.13) IL6ST 167 0.774 1.05 (0.77, 1.43) 0.541 1.12 (0.77, 1.63) 0.235 0.81 (0.57, 1.15) INHBA 167 0.104 1.30 (0.95, 1.78) 0.002 1.89 (1.26, 2.84) 0.077 1.38 (0.97, 1.97) ITGA7 167 0.990 1.00 (0.73, 1.36) 0.780 1.05 (0.73, 1.53) 0.470 0.88 (0.62, 1.25) JUN 167 0.586 1.09 (0.80, 1.48) 0.538 0.89 (0.62, 1.28) 0.259 0.82 (0.59, 1.15) KLK2 167 0.267 0.84 (0.61, 1.15) 0.003 0.56 (0.38, 0.82) 0.007 0.61 (0.42, 0.87) KRT15 167 0.500 0.90 (0.65, 1.23) 0.738 0.94 (0.65, 1.35) 0.987 1.00 (0.71, 1.42) KRT5 152 0.834 0.97 (0.70, 1.34) 0.632 1.10 (0.74, 1.63) 0.908 0.98 (0.68, 1.40) LAMB3 167 0.090 1.31 (0.96, 1.79) 0.013 1.73 (1.12, 2.68) 0.132 1.33 (0.92, 1.94) LGALS3 166 0.345 1.16 (0.85, 1.59) 0.405 1.18 (0.80, 1.72) 0.208 0.80 (0.57, 1.13) MMP11 167 0.715 1.06 (0.78, 1.45) 0.080 1.37 (0.96, 1.96) 0.257 1.22 (0.87, 1.71) MYBL2 167 0.235 1.21 (0.88, 1.67) 0.868 1.03 (0.71, 1.49) 0.266 1.21 (0.86, 1.70) NFAT5 167 0.514 0.90 (0.66, 1.23) 0.058 0.70 (0.48, 1.01) 0.530 0.89 (0.63, 1.27) OLFML3 167 0.448 0.89 (0.65, 1.21) 0.056 1.50 (0.99, 2.28) 0.129 0.77 (0.54, 1.08) PAGE4 167 0.914 0.98 (0.72, 1.34) 0.211 0.80 (0.56, 1.14) 0.005 0.61 (0.43, 0.86) PGK1 167 0.138 0.78 (0.56, 1.08) 0.666 0.92 (0.64, 1.33) 0.292 0.83 (0.59, 1.17) PPAP2B 167 0.952 0.99 (0.73, 1.35) 0.989 1.00 (0.69, 1.44) 0.221 0.80 (0.56, 1.14) PPP1R12A 167 0.547 0.91 (0.66, 1.24) 0.563 0.90 (0.63, 1.29) 0.001 0.55 (0.38, 0.79) PRKCA 167 0.337 1.17 (0.85, 1.59) 0.141 1.35 (0.90, 2.03) 0.029 0.67 (0.46, 0.96) SDC1 167 0.064 1.36 (0.98, 1.87) 0.013 1.83 (1.14, 2.96) 0.037 1.58 (1.03, 2.42) SFRP4 166 0.986 1.00 (0.73, 1.37) 0.047 1.47 (1.01, 2.15) 0.031 1.49 (1.04, 2.14) SHMT2 167 0.133 0.78 (0.56, 1.08) 0.147 0.77 (0.53, 1.10) 0.715 0.94 (0.66, 1.33) SLC22A3 167 0.828 1.03 (0.76, 1.41) 0.044 0.69 (0.48, 0.99) 0.050 0.71 (0.50, 1.00) SMAD4 167 0.165 1.25 (0.91, 1.71) 0.333 0.83 (0.58, 1.21) 0.021 0.65 (0.45, 0.94) SPARC 167 0.810 0.96 (0.71, 1.31) 0.000 2.15 (1.40, 3.30) 0.154 1.30 (0.91, 1.86) SRC 167 0.083 1.34 (0.96, 1.86) 0.750 1.06 (0.72, 1.56) 0.550 0.90 (0.64, 1.26) SRD5A2 167 0.862 0.97 (0.71, 1.33) 0.122 0.75 (0.53, 1.08) 0.010 0.63 (0.45, 0.90) STAT5B 167 0.298 0.84 (0.62, 1.16) 0.515 0.89 (0.62, 1.27) 0.016 0.65 (0.46, 0.92) TGFB1I1 167 0.985 1.00 (0.74, 1.37) 0.066 1.45 (0.98, 2.14) 0.131 0.76 (0.54, 1.08) THBS2 167 0.415 1.14 (0.83, 1.56) 0.001 1.91 (1.30, 2.80) 0.288 1.21 (0.85, 1.70) TNFRSF10B 167 0.214 1.22 (0.89, 1.66) 0.805 0.95 (0.66, 1.38) 0.118 0.76 (0.54, 1.07) TPM2 167 0.996 1.00 (0.73, 1.36) 0.527 1.13 (0.78, 1.64) 0.094 0.74 (0.52, 1.05) TPX2 167 0.017 1.48 (1.07, 2.04) 0.002 1.89 (1.26, 2.83) 0.001 1.91 (1.30, 2.80) TUBB2A 167 0.941 0.99 (0.73, 1.35) 0.182 0.78 (0.54, 1.12) 0.111 0.75 (0.53, 1.07) UBE2T 167 0.095 1.36 (0.95, 1.96) 0.009 1.58 (1.12, 2.23) 0.084 1.33 (0.96, 1.84) VCL 167 0.954 0.99 (0.73, 1.35) 0.165 1.31 (0.90, 1.91) 0.265 0.82 (0.57, 1.16) ZFP36 167 0.685 1.07 (0.78, 1.45) 0.784 0.95 (0.66, 1.37) 0.610 0.91 (0.64, 1.29)

TABLE 4 RS Model ECM (Stromal Response) Migration (Cellular Organization) Prolif. Androgen (PSA) Other Algorithm RS0 (ASPN + (FLNC + GSN + GSTM2 + (TPX2 + (FAM13C + KLK2)/2 STAT5B, NFAT5 1.05 * ECM − 0.58 * Migration − BGN + COL1A1 + SPARC)/4 IGFBP6 + PPAP2B + PPP1R12A)/6 CDC20 + 0.30 * PSA + 0.08 * Prolif − MYBL2)/3 0.16 * STAT5B − 0.23 * NFAT5 RS1 (BGN + (FLNC + GSN + GSTM2 + PPAP2B + — (FAM13C + KLK2)/2 STAT5B, NFAT5 1.15 * ECM − 0.72 * Migration − COL1A1 + FN1 + SPARC)/4 PPP1R12A)/6 0.56 * PSA − 0.45 * STAT5B − 0.56 * NFAT5 RS2 (BGN + COL1A1 + FN1 + (BIN1 + FLNC + GSN + GSTM2 + — (FAM13C + KLK2)/2 STAT5B, NFAT5 1.16 * ECM − 0.75 * Migration − SPARC)/4 PPAP2B + PPP1R12A + VCL)/7 0.57 * PSA − 0.47 * STAT5B − 0.50 * NFAT5 RS3 (BGN + COL1A1 + COL3A1 + (FLNC + GSN + GSTM2 + PPAP2B + — (FAM13C + KLK2)/2 STAT5B, NFAT5 1.18 * ECM − 0.75 * Migration − COL4A1 + FN1 + SPARC)/6 PPP1R12A)/5 0.56 * PSA − 0.40 * STAT5B − 0.48 * NFAT5 RS4 (BGN + COL1A1 + COL3A1 + (BIN1 + FLNC + GSN + GSTM2 + — (FAM13C + KLK2)/2 1.18 * ECM − 0.76 * Migration − COL4A1 + FN1 + SPARC)/6 PPAP2B + PP1R12A + VCL)/7 0.58 * PSA − 0.43 * STAT5B − 0.43 * NFAT5 RS5 (COL4A1 (thresholded) + (BIN1 + IGF1 (thresholded) + — KLK2 AZGP1, ANPEP, 1.20 * ECM − 0.91 * Migration − INHBA + SPARC + THBS2)/4 VCL)/3 IGFBP2 0.29 * KLK2 − (thresholded) 0.14 * AZGP1 + 0.05 * ANPEP − 0.56 * IGFBP2 RS6 (BGN + COL3A1 + INHBA + Migratn1: (FLNC + GSN + TPM2)/3 TPX2 (FAM13C + KLK2)/2 AZGP1, SLC22A3 1.09 * ECM − 0.44 * Migration1 − SPARC)/4 Migratn2: (GSTM2 + PPAP2B)/2 0.23 * Migratn2 − 0.36 * PSA + 0.15 * TPX2 − 0.16 * AZGP1 − 0.08 * SLC22A3 RS7 (BGN + COL3A1 + INHBA + Migratn1: (FLNC + GSN + TPM2)/3 — (FAM13C + KLK2)/2 AZGP1, SLC22A3 1.16 * ECM − 0.53 * Migration1 − SPARC)/4 Migratn2: (GSTM2 + PPAP2B)/2 0.24 * Migratn2 − 0.42 * PSA − 0.14 * AZGP1 − 0.08 * SLC22A3 RS8 (BGN + COL3A1 + SPARC)/3 Migratn1: (FLNC + GSN + TPM2)/3 — KLK2 AZGP1, SLC22A3 1.37 * ECM − 0.56 * Migration1 − Migratn2: (GSTM2 + PPAP2B)/2 0.49 * Migratn2 − 0.52 * KLK2 − 0.16 * AZGP1 − 0.00 * SLC22A3 RS9 (BGN Migratn1: (FLNC (thresholded) + — (FAM13C + KLK2)/2 AZGP1, SLC22A3 1.28 * ECM − 1.11 * Migration1 − (thresholded) + COL3A1 + GSN (thresholded) + TPM2)/3 0.00 * Migratn2 − 0.34 * PSA − INHBA + SPARC Migratn2: (GSTM2 + PPAP2B)/2 0.16 * AZGP1 − 0.08 * SLC22A3 (thresholded))/4 RS10 (BGN + COL3A1 + INHBA + (FLNC + GSN + GSTM2 + PPAP2B + TPX2 (FAM13C + KLK2)/2 AZGP1, SLC22A3 1.09 * ECM − 0.68 * Migration − SPARC)/4 TPM2)/5 0.37 * PSA + 0.16 * TPX2 − 0.16 * AZGP1 − 0.08 * SLC22A3 RS11 (BGN (thresholded) + (FLNC(thresholded) + — (FAM13C + KLK2)/2 AZGP1, SLC22A3 1.19 * ECM − 0.96 * Migration − COL3A1 + INHBA + GSN(thresholded) + GSTM2 + 0.39 * PSA − 0.14 * AZGP1 − SPARC(thresholded))/4 PPAP2B + TPM2)/5 0.09 * SLC22A3 RS12 (BGN (thresholded) + (FLNC(thresholded) + TPX2 (FAM13C + KLK2)/2 AZGP1, SLC22A3 1.13 * ECM − 0.85 * Migration − COL3A1 + INHBA + GSN(thresholded) + GSTM2 + 0.34 * PSA + 0.15 * TPX2 − SPARC(thresholded))/4 PPAP2B + TPM2)/5 0.15 * AZGP1 − 0.08 * SLC22A3 RS13 (BGN (thresholded) + (FLNC(thresholded) + TPX2 (FAM13C + KLK2)/2 AZGP1, ERG, 1.12 * ECM − 0.83 * Migratn − COL3A1 + INHBA + GSN(thresholded) + GSTM2 + SLC22A3 0.33 * PSA + 0.17 * TPX2 − SPARC(thresholded))/4 PPAP2B + TPM2)/5 0.14 * AZGP1 + 0.04 * ERG − 0.10 * SLC22A3 RS14 (BGN (thresholded) + (FLNC(thresholded) + TPX2 (FAM13C + KLK2)/2 AR, AZGP1, ERG, 1.13 * ECM − 0.83 * Migration − COL3A1 + INHBA + GSN(thresholded) + GSTM2 + SLC22A3 0.35 * PSA + 0.16 * TPX2 + 0.15 * AR − SPARC(thresholded))/4 PPAP2B + TPM2)/5 0.15 * AZGP1 + 0.03 * ERG − 0.10 * SLC22A3 RS15 (BGN (thresholded) + (FLNC(thresholded) + — KLK2 AR, ERG, SLC22A3 1.30 * ECM − 1.20 * Migration − COL3A1 + INHBA + GSN(thresholded) + GSTM2 + 0.52 * KLK2 + 0.09 * AR + 0.05 * ERG − SPARC(thresholded))/4 PPAP2B + TPM2)/5 0.06 * SLC22A3 RS16 (BGN (thresholded) + (C7 + FLNC(thresholded) + — KLK2 AR, ERG, SLC22A3 1.23 * ECM − 1.02 * Migration − COL3A1 + INHBA + GSN(thresholded) + GSTM1)/4 0.46 * KLK2 + 0.09 * AR + 0.07 * ERG − SPARC(thresholded))/4 0.09 * SLC22A3 RS17 (BGN + COL1A1 + SFRP4)/3 (FLNC + GSN + GSTM1 + TPM2)/4 TPX2 (FAM13C + KLK2)/2 AR, AZGP1, ERG, 0.63 * ECM − 0.12 * Migration − SLC22A3, SRD5A2 0.44 * PSA + 0.19 * TPX2 − 0.02 * AR − 0.15 * AZGP1 + 0.06 * ERG − 0.13 * SLC22A3 − 0.33 * SRD5A2 RS18 (BGN + COL1A1 + SFRP4)/3 (FLNC + GSN + GSTM1 + TPM2)/4 TPX2 (FAM13C + KLK2)/2 AR, ERG, 0.63 * ECM − 0.17 * Migration4 − SLC22A3, SRD5A2 0.52 * PSA + 0.19 * TPX2 − 0.07 * AR + 0.09 * ERG − 0.14 * SLC22A3 − 0.36 * SRD5A2 RS19 (BGN + COL1A1 + SFRP4)/3 (FLNC + GSN + GSTM1 + TPM2)/4 — (FAM13C + KLK2)/2 AR, AZGP1, ERG, 0.72 * ECM − 0.24 * Migration4 − SLC22A3, SRD5A2 0.51 * PSA + 0.03 * AR − 0.15 * AZGP1 + 0.04 * ERG − 0.12 * SLC22A3 − 0.32 * SRD5A2 RS20 (BGN + COL1A1 + SFRP4)/3 (FLNC + GSN + PPAP2B + TPM2)/4 TPX2 (FAM13C + KLK2)/2 (Stress: 0.72 * ECM − 0.26 * Migration − GSTM1 + GSTM2) 0.45 * PSA + 0.15 * TPX2 + AZGP1, SLC22A3, 0.02 * Stress − 0.16 * AZGP1 − SRD5A2 0.06 * SLC22A3 − 0.30 * SRD5A2 RS21 (BGN + COL1A1 + SFRP4)/3 (FLNC + GSN + PPAP2B + TPM2)/4 TPX2 (FAM13C + KLK2)/2 AZGP1, SLC22A3, 0.68 * ECM − 0.19 * Migration − SRD5A2 0.43 * PSA + 0.16 * TPX2 − 0.18 * AZGP1 − 0.07 * SLC22A3 − 0.31 * SRD5A2 RS22 (BGN + COL1A1 + SFRP4)/3 TPX2 (FAM13C + KLK2)/2 (Stress: 0.62 * ECM − 0.46 * PSA + GSTM1 + GSTM2) 0.18 * TPX2 − 0.07 * Stress − AZGP1, SLC22A3, 0.18 * AZGP1 − 0.08 * SLC22A3 − SRD5A2 0.34 * SRD5A2 RS23 (BGN + COL1A1 + SFRP4)/3 (FLNC + GSN + GSTM2 + TPM2)/4 TPX2 (FAM13C + KLK2)/2 AR, AZGP1, ERG, 0.73 * ECM − 0.26 * Migration − SRD5A2 0.45 * PSA + 0.17 * TPX2 + 0.02 * AR − 0.17 * AZGP1 + 0.03 * ERG − 0.29 * SRD5A2 RS24 (BGN + COL1A1 + SFRP4)/3 (FLNC + GSN + GSTM1 + GSTM2 + TPX2 (FAM13C + KLK2)/2 AZGP1, SLC22A3, 0.52 * ECM − 0.23 * Migration − PPAP2B + TPM2)/6 SRD5A2 0.30 * PSA + 0.14 * TPX2 − 0.17 * AZGP1 − 0.07 * SLC22A3 − 0.27 * SRD5A2 RS25 (BGN + COL1A1 + SFRP4)/3 (FLNC + GSN + TPM2)/3 TPX2 (FAM13C + KLK2)/2 AZGP1, GSTM2, 0.72 * ECM − 0.14 * Migration − SRD5A2 0.45 * PSA + 0.16 * TPX2 − 0.17 * AZGP11 − 0.14 * GSTM2 − 0.28 * SRD5A2 RS26 (1.581 * BGN + 1.371 * COL1A1 + (0.489 * FLNC + 1.512 * GSN + 1.264 * TPX2 (1.267 * FAM13C + AZGP1, GSTM2, 0.735 * ECM − 0.368 * Migration − 0.469 * SFRP4)/3 TPM2)/3 (thresholded) 2.158 * KLK2)/2 SRD5A2 0.352 * PSA + 0.094 * TPX2 − (thresholded) 0.226 * AZGP11 − 0.145 * GSTM2 − 0.351 * SRD5A2 RS27 (1.581 * BGN + 1.371 * COL1A1 + [(0.489 * FLNC + 1.512 * GSN + 1.264 * TPX2 [(1.267 * FAM13C + 0.735 * ECM − 0.368 * Migration − 0.469 * SFRP4)/3 = TPM2)/3] + (0.145 * GSTM2/0.368) = (thresholded) 2.158 * KLK2)/2] + 0.352 * PSA + 0.095 * TPX2 0.527 * BGN + 0.163 * FLNC + 0.504 * GSN + (0.226 * AZGP1/0.352) + 0.457 * COL1A1 + 0.421 * TPM2 + 0.394 * GSTM2 (0.351 * SRD5A2Thresh/ 0.156 * SFRP4 0.352) = 0.634 * FAM13C + 1.079 * KLK2 + 0.642 * AZGP1 + 0.997SRD5A2Thresh

Table 5A shows the standardized odds ratio of each of the RS models using the data from the original Gene ID study described in Example 1 for time to cR and for upgrading and upstaging and the combination of significant upgrading and upstaging. Table 5B shows the performance of each of the RS models using the data from the CC Companion (Cohorts 2 and 3) study for upgrading and upstaging and the combination of significant upgrading and upstaging. In this context, “upgrading” refers to an increase in Gleason grade from 3+3 or 3+4 at biopsy to greater than or equal to 3+4 at radical prostatectomy. “Significant upgrading” in this context refers to upgrading from Gleason grade 3+3 or 3+4 at biopsy to equal to or greater than 4+3 at radical prostatectomy.

In addition, the gene groups used in the RS25 model were evaluated alone and in various combinations. Table 6A shows the results of this analysis using the data from the Gene Identification study and Table 6B shows the results of this analysis using the data from Cohorts 2 and 3 of the CC Companion Study.

The gene expression for some genes may be thresholded, for example SRD5A2 Thresh=5.5 if SRD5A2<5.5 or SRD5A2 if SRD5A2≥ 5.5 and TPX2 Thresh=5.0 if TPX2<5.0 or TPX2 if TPX2≥ 5.0, wherein the gene symbols represent normalized gene expression values.

The unscaled RS scores derived from Table 4 can also be rescaled to be between 0 and 100. For example, RS27 can be rescaled to be between 0 and 100 as follows:

RS (scaled)=0 if 13.4×(RSu+10.5)<0; 13.4×(RSu+10.5) if 0≤13.4×(RSu+10.5)≤100; or 100 if 13.4×(RSu+10.5)>100.

Using the scaled RS, patients can be classified into low, intermediate, and high RS groups using pre-specified cut-points defined below in Table B. These cut-points define the boundaries between low and intermediate RS groups and between intermediate and high RS groups. The cutpoints were derived from the discovery study with the intent of identifying substantial proportions of patients who on average had clinically meaningful low or high risk of aggressive disease. The scaled RS is rounded to the nearest integer before the cut-points defining RS groups are applied.

TABLE B RS Group Risk Score Low Less than 16 Intermediate Greater than or equal to 16 and less than 30 High Greater than or equal to 30

TABLE 5A Significant Significant Upgrading Upgrading Upgrading Upstaging or Upstaging RS N OR 95% CI OR 95% CI OR 95% CI OR 95% CI RS0 280 1.72 (1.22, 2.41) 7.51 (4.37, 12.9) 2.01 (1.41, 2.88) 2.91 (1.95, 4.34) RS1 287 1.73 (1.21, 2.48) 5.98 (3.30, 10.8) 1.99 (1.40, 2.82) 2.68 (1.80, 3.97) RS2 287 1.72 (1.19, 2.48) 5.89 (3.18, 10.9) 2.02 (1.42, 2.86) 2.67 (1.80, 3.95) RS3 287 1.71 (1.20, 2.45) 6.30 (3.66, 10.8) 1.96 (1.38, 2.80) 2.69 (1.84, 3.93) RS4 287 1.69 (1.18, 2.42) 6.06 (3.48, 10.5) 1.99 (1.40, 2.82) 2.65 (1.82, 3.86) RS5 288 1.78 (1.21, 2.62) 5.60 (3.56, 8.81) 2.24 (1.59, 3.15) 2.87 (1.93, 4.28) RS6 287 1.94 (1.37, 2.74) 10.16 (5.82, 17.8) 2.07 (1.48, 2.91) 3.11 (2.07, 4.67) RS7 288 1.91 (1.34, 2.71) 9.34 (5.25, 16.6) 2.06 (1.47, 2.89) 3.01 (2.02, 4.48) RS8 289 1.80 (1.27, 2.55) 7.49 (4.02, 14.0) 2.09 (1.49, 2.92) 2.86 (1.97, 4.14) RS9 288 2.00 (1.39, 2.89) 9.56 (5.06, 18.0) 1.99 (1.42, 2.79) 3.09 (2.08, 4.60) RS10 287 1.94 (1.37, 2.75) 10.12 (5.79, 17.7) 2.09 (1.49, 2.94) 3.14 (2.08, 4.74) RS11 288 2.09 (1.43, 3.05) 9.46 (5.18, 17.3) 2.17 (1.54, 3.05) 3.42 (2.24, 5.23) RS12 287 2.10 (1.45, 3.04) 10.41 (5.92, 18.3) 2.17 (1.55, 3.06) 3.52 (2.30, 5.40) RS13 287 2.10 (1.44, 3.05) 9.40 (5.50, 16.1) 2.20 (1.55, 3.13) 3.50 (2.25, 5.43) RS14 287 2.06 (1.42, 2.99) 9.71 (5.65, 16.7) 2.18 (1.55, 3.08) 3.53 (2.29, 5.44) RS15 288 1.92 (1.32, 2.78) 7.93 (4.56, 13.8) 2.12 (1.51, 2.99) 3.25 (2.20, 4.80) RS16 288 1.76 (1.23, 2.52) 7.10 (4.12, 12.2) 1.99 (1.41, 2.82) 2.94 (1.98, 4.38) RS17 286 2.23 (1.52, 3.27) 7.52 (4.18, 13.5) 2.91 (1.93, 4.38) 4.48 (2.72, 7.38) RS18 286 2.12 (1.46, 3.08) 7.04 (3.87, 12.8) 2.89 (1.91, 4.37) 4.30 (2.62, 7.06) RS19 287 2.14 (1.46, 3.13) 6.90 (3.80, 12.5) 2.88 (1.96, 4.23) 4.20 (2.66, 6.63) RS20 286 2.30 (1.55, 3.42) 8.41 (4.65, 15.2) 2.90 (1.98, 4.25) 4.78 (3.00, 7.61) RS21 287 2.36 (1.59, 3.52) 8.83 (4.87, 16.0) 2.63 (1.76, 3.94) 4.93 (3.06, 7.93) RS22 286 2.16 (1.48, 3.15) 7.57 (4.14, 13.8) 2.90 (1.96, 4.27) 4.39 (2.75, 7.01) RS23 287 2.26 (1.53, 3.35) 7.46 (4.24, 13.1) 2.80 (1.85, 4.24) 4.79 (2.98, 7.68) RS24 286 2.21 (1.50, 3.24) 8.01 (4.38, 14.7) 2.93 (1.99, 4.31) 4.62 (2.89, 7.39) RS25 287 2.25 (1.53, 3.31) 7.70 (4.25, 14.0) 2.76 (1.83, 4.16) 4.76 (2.99, 7.58) RS26 287 2.23 (1.51, 3.29) 6.67 (3.52, 12.7) 2.64 (1.81, 3.86) 4.01 (2.56, 6.28) RS27 287 2.23 (1.51, 3.29) 6.67 (3.52, 12.7) 2.64 (1.81, 3.86) 4.01 (2.56, 6.28)

TABLE 5B Significant Significant Upgrading Upgrading Upgrading Upstaging or Upstaging Model N Std OR 95% CI Std OR 95% CI Std OR 95% CI Std OR 95% CI RS0 166 1.16 (0.84, 1.58) 2.45 (1.61, 3.73) 2.42 (1.61, 3.62) 3 (1.98, 4.56) RS1 167 1.05 (0.77, 1.43) 2.46 (1.63, 3.71) 2.38 (1.61, 3.53) 3.36 (2.18, 5.18) RS2 167 1.04 (0.76, 1.42) 2.45 (1.63, 3.69) 2.34 (1.58, 3.46) 3.25 (2.12, 4.99) RS3 167 1.04 (0.76, 1.41) 2.56 (1.69, 3.89) 2.28 (1.55, 3.36) 3.27 (2.13, 5.03) RS4 167 1.03 (0.75, 1.40) 2.54 (1.68, 3.86) 2.23 (1.52, 3.27) 3.16 (2.07, 4.82) RS5 167 1.02 (0.75, 1.39) 1.89 (1.28, 2.78) 1.77 (1.23, 2.55) 2.21 (1.52, 3.20) RS6 167 1.08 (0.79, 1.48) 2.49 (1.64, 3.79) 2.42 (1.62, 3.62) 3.22 (2.09, 4.96) RS7 167 1.03 (0.75, 1.40) 2.31 (1.54, 3.48) 2.28 (1.54, 3.38) 2.97 (1.96, 4.51) RS8 167 0.94 (0.69, 1.28) 2.34 (1.55, 3.53) 2.31 (1.56, 3.43) 2.87 (1.91, 4.30) RS9 167 1.02 (0.75, 1.39) 2.19 (1.47, 3.27) 2.22 (1.51, 3.27) 2.77 (1.85, 4.14) RS10 167 1.08 (0.79, 1.48) 2.49 (1.63, 3.78) 2.41 (1.61, 3.61) 3.22 (2.09, 4.95) RS11 167 0.99 (0.73, 1.35) 2.18 (1.46, 3.24) 2.17 (1.48, 3.19) 2.83 (1.88, 4.25) RS12 167 1.06 (0.78, 1.45) 2.36 (1.57, 3.56) 2.34 (1.57, 3.48) 3.12 (2.04, 4.78) RS13 166 1.01 (0.74, 1.37) 2.17 (1.45, 3.23) 2.41 (1.61, 3.60) 2.99 (1.97, 4.54) RS14 166 1.03 (0.76, 1.41) 2.22 (1.48, 3.31) 2.33 (1.57, 3.46) 2.95 (1.94, 4.47) RS15 166 1 (0.73, 1.36) 1.98 (1.34, 2.92) 2.12 (1.44, 3.12) 2.58 (1.74, 3.84) RS16 166 0.94 (0.69, 1.28) 1.7 (1.16, 2.48) 2.07 (1.41, 3.03) 2.24 (1.54, 3.25) RS17 165 0.98 (0.72, 1.34) 1.96 (1.33, 2.89) 2.63 (1.73, 3.98) 3.02 (1.99, 4.60) RS18 165 0.97 (0.71, 1.33) 1.86 (1.26, 2.73) 2.71 (1.78, 4.13) 3.01 (1.98, 4.56) RS19 165 0.93 (0.68, 1.27) 1.86 (1.27, 2.72) 2.4 (1.61, 3.58) 2.75 (1.84, 4.10) RS20 166 1.07 (0.78, 1.46) 2.2 (1.48, 3.29) 2.47 (1.65, 3.69) 3.1 (2.04, 4.72) RS21 166 1.06 (0.77, 1.45) 2.2 (1.47, 3.28) 2.48 (1.65, 3.71) 3.11 (2.04, 4.74) RS22 166 1.04 (0.76, 1.43) 2.21 (1.48, 3.29) 2.47 (1.65, 3.70) 3.14 (2.05, 4.79) RS23 165 1.02 (0.75, 1.40) 2.01 (1.36, 2.97) 2.52 (1.67, 3.79) 2.94 (1.95, 4.44) RS24 166 1.04 (0.76, 1.42) 2.18 (1.46, 3.26) 2.52 (1.68, 3.78) 3.14 (2.06, 4.80) RS25 166 1.04 (0.76, 1.42) 2.11 (1.42, 3.13) 2.45 (1.64, 3.67) 3 (1.98, 4.54) RS26 166 0.99 (0.72, 1.35) 2.05 (1.38, 3.04) 2.43 (1.63, 3.65) 2.82 (1.88, 4.21) RS27 166 0.99 (0.72, 1.35) 2.05 (1.38, 3.04) 2.43 (1.63, 3.65) 2.82 (1.88, 4.21)

TABLE 6A Significant Significant Upgrading or Time to cR Upgrading Upgrading Upstaging Upstaging Model N Std HR N Std OR 95% CI Std OR 95% CI Std OR 95% CI Std OR 95% CI RS25 428 2.82 232 2.09 (1.41, 3.10) 7.35 (3.87, 14.0) 2.55 (1.63, 4.00) 4.46 (2.72, 7.32) Stromal 430 2.05 234 1.32 (0.95, 1.84) 3.08 (1.84, 5.14) 1.6 (1.12, 2.30) 1.95 (1.35, 2.82) Cellular Organization 430 1.67 234 1.67 (1.16, 2.39) 2.83 (1.63, 4.90) 1.38 (0.96, 1.99) 2.06 (1.37, 3.10) PSA 430 1.89 234 0.96 (0.70, 1.32) 1.38 (0.72, 2.63) 1.47 (1.06, 2.03) 1.25 (0.83, 1.88) ECM Cellular Organization 430 2.6 234 2 (1.37, 2.93) 11.5 (5.84, 22.7) 1.98 (1.34, 2.93) 4.01 (2.44, 6.58) ECM PSA 430 2.45 234 1.17 (0.85, 1.61) 2.46 (1.44, 4.21) 1.7 (1.21, 2.39) 1.76 (1.22, 2.53) Cellular Organization PSA 430 2.04 234 1.3 (0.92, 1.82) 2.52 (1.23, 5.16) 1.63 (1.13, 2.36) 1.85 (1.19, 2.87) ECM Cellular Organization 429 2.61 233 1.89 (1.31, 2.72) 11.3 (5.46, 23.5) 1.94 (1.31, 2.87) 3.99 (2.44, 6.54) TPX2 ECM PSA TPX2 429 2.42 233 1.24 (0.90, 1.71) 3.25 (1.91, 5.51) 1.75 (1.22, 2.49) 2.08 (1.45, 2.98) Cellular Organization PSA 429 2.04 233 1.33 (0.95, 1.86) 3.2 (1.74, 5.90) 1.69 (1.17, 2.44) 2.21 (1.45, 3.37) TPX2 ECM Cellular Organization 430 2.67 234 2.03 (1.39, 2.96) 11.3 (5.72, 22.3) 2.17 (1.43, 3.30) 4.35 (2.50, 7.58) GSTM2 ECM PSA GSTM2 430 2.86 234 1.48 (1.05, 2.09) 4.45 (2.03, 9.76) 2.2 (1.45, 3.34) 2.66 (1.64, 4.31) Cellular Organization PSA 430 2.25 234 1.34 (0.94, 1.90) 2.52 (1.18, 5.38) 1.92 (1.29, 2.84) 2.02 (1.20, 3.39) GSTM2 ECM Cellular Organization 428 2.72 232 2.38 (1.58, 3.57) 11.5 (6.02, 21.8) 2.48 (1.58, 3.87) 5.22 (2.97, 9.17) GSTM2 TPX2 AZGP1 SRD5A2 ECM PSA GSTM2 TPX2 428 2.8 232 2.03 (1.38, 3.00) 6.65 (3.52, 12.6) 2.6 (1.65, 4.09) 4.26 (2.58, 7.02) AZGP1 SRD5A2 Cellular Organization PSA 428 2.38 232 1.92 (1.28, 2.88) 3.63 (2.14, 6.15) 2.6 (1.64, 4.12) 3.49 (2.08, 5.83) GSTM2 TPX2 AZGP1 SRD5A2

TABLE 6B Significant Significant Upgrading or Upgrading Upgrading Upstaging Upstaging Model N Std OR 95% CI Std OR 95% CI Std OR 95% CI Std OR 95% CI RS25 166 1.04 (0.76, 1.42) 2.11 (1.42, 3.13) 2.45 (1.64, 3.67) 3 (1.98, 4.54) Stromal 166 0.99 (0.73, 1.35) 2.19 (1.45, 3.32) 1.65 (1.15, 2.38) 1.86 (1.31, 2.65) Cellular Organization 167 1.06 (0.77, 1.44) 0.93 (0.64, 1.36) 1.49 (1.04, 2.13) 1.44 (1.03, 2.00) PSA 167 1.04 (0.76, 1.42) 1.68 (1.16, 2.44) 1.78 (1.24, 2.57) 1.96 (1.37, 2.81) ECM Cellular Organization 166 1.04 (0.76, 1.42) 1.96 (1.32, 2.91) 2.32 (1.55, 3.45) 2.6 (1.76, 3.85) ECM PSA 166 1.02 (0.75, 1.39) 2.14 (1.44, 3.20) 1.84 (1.28, 2.67) 2.11 (1.47, 3.04) Cellular Organization PSA 167 1.07 (0.78, 1.46) 1.36 (0.94, 1.97) 2.06 (1.40, 3.04) 2.12 (1.47, 3.06) ECM Cellular Organization 166 1.15 (0.84, 1.58) 2.24 (1.49, 3.37) 2.55 (1.69, 3.85) 2.95 (1.96, 4.45) TPX2 ECM PSA TPX2 166 1.2 (0.88, 1.65) 2.66 (1.71, 4.13) 2.28 (1.53, 3.40) 2.72 (1.82, 4.07) Cellular Organization PSA 167 1.3 (0.95, 1.79) 1.77 (1.21, 2.60) 2.42 (1.62, 3.63) 2.65 (1.79, 3.92) TPX2 ECM Cellular Organization 166 0.96 (0.70, 1.30) 1.76 (1.20, 2.57) 2.12 (1.44, 3.12) 2.34 (1.60, 3.42) GSTM2 ECM PSA GSTM2 166 0.91 (0.67, 1.24) 1.69 (1.16, 2.46) 1.85 (1.28, 2.67) 2.05 (1.42, 2.94) Cellular Organization PSA 167 0.89 (0.65, 1.22) 1.13 (0.78, 1.62) 1.72 (1.19, 2.48) 1.75 (1.23, 2.48) GSTM2 ECM Cellular Organization 166 1.04 (0.76, 1.42) 2.14 (1.44, 3.20) 2.47 (1.65, 3.70) 2.94 (1.95, 4.44) GSTM2 TPX2 AZGP1 SRD5A2 ECM PSA GSTM2 TPX2 AZGP1 166 1.03 (0.75, 1.41) 2.11 (1.42, 3.13) 2.39 (1.61, 3.57) 2.94 (1.95, 4.45) SRD5A2 Cellular Organization 167 1.07 (0.78, 1.46) 1.84 (1.26, 2.68) 2.22 (1.51, 3.27) 2.73 (1.83, 4.09) PSA GSTM2 TPX2 AZGP1 SRD5A2

Example 3: Clique Stack Analysis to Identify Co-Expressed Genes

The purpose of the gene clique stacks method described in this Example was to find a set of co-expressed (or surrogate) biomarkers that can be used to reliably predict outcome as well or better than the genes disclosed above. The method used to identify the co-expressed markers is illustrated in FIG. 4. The set of co-expressed biomarkers were obtained by seeding the maximal clique enumeration (MCE) with curated biomarkers extracted from the scientific literature. The maximal clique enumeration (MCE) method [Bron et al, 1973] aggregates genes into tightly co-expressed groups such that all of the genes in the group have a similar expression profile. When all of the genes in a group satisfy a minimal similarity condition, the group is called a clique. When a clique is as large as possible without admitting any ‘dissimilar’ genes into the clique, then the clique is said to be maximal. Using the MCE method, all maximal cliques are searched within a dataset. Using this method, almost any degree of overlap between the maximal cliques can be found, as long as the overlap is supported by the data. Maximal clique enumeration has been shown [Borate et al, 2009] to be an effective way of identifying co-expressed gene modules (CGMs).

1. Definitions

The following table defines a few terms commonly used in the gene clique stack analyses.

TABLE 7 Term Definition Node The abundance of a gene (for the purposes of CGM analysis) Edge A line connecting two nodes, indicating co-expression of the two nodes Graph A collection of nodes and edges Clique A graph with an edge connecting all pair-wise combinations of nodes in the graph maximal A clique that is not contained in any other clique clique Stack A graph obtained by merging at least two cliques or stacks such that the overlap between the two cliques or stacks exceeds some user-defined threshold. gene A two-dimensional matrix, with genes listed down the expression rows and samples listed across the columns. Each (i, j) profile entry in the matrix corresponds to relative mRNA abundance for gene i and sample j.

2. Examples of Cliques and Stacks

FIG. 5 shows a family of three different graphs. A graph consists of nodes (numbered) and connecting edges (lines). FIG. 5(a) is not a clique because there is no edge connecting nodes 3 and 4. FIG. 5(b) is a clique because there is an edge connecting all pair-wise combinations of nodes in the graph. FIG. 5(c) is a clique, but not a maximal clique because it is contained in clique (b). Given a graph with connecting edges, the MCE algorithm will systematically list all of maximal cliques with 3 or more nodes. For example, the graph in FIG. 6 has two maximal cliques: 1-2-3-4-5 and 1-2-3-4-6.

When based on gene expression data, there are typically large numbers of maximal cliques that are very similar to one another. These maximal cliques can be merged into stacks of maximal cliques. The stacks are the final gene modules of interest and generally are far fewer in number than are the maximal cliques. FIG. 7A-C schematically illustrates stacking of two maximal cliques.

3. Seeding

For the purposes of finding surrogate co-expressed markers, biomarkers from the literature can be identified and then used to seed the MCE and stacking algorithms. The basic idea is as follows: for each seed, compute a set of maximal cliques (using the parallel MCE algorithm). Then stack the maximal cliques obtained for each seed, yielding a set of seeded stacks. Finally, stack the seeded stacks to obtain a “stack of seeded stacks.” The stack of seeded stacks is an approximation to the stacks that would be obtained by using the conventional (i.e. unseeded) MCE/stacking algorithms. The method used to identify genes that co-express with the genes disclosed above illustrated in FIG. 4 and is described in more detail below.

3.1 Seeded MCE Algorithm (Steps 1-4)

1. The process begins by identifying an appropriate set, S_(s), of seeding genes. In the instant case, the seeding genes were selected from the gene subsets disclosed above.

2. With the seeding genes specified, select a measure of correlation, R(g₁,g₂), between the gene expression profiles of any two genes, g₁,g₂, along with a correlation threshold below which g₁,g₂, can be considered uncorrelated. For each seeding gene s in the seeding set S_(s), find all gene pairs (s,g) in the dataset such that R(s,g) is greater than or equal to the correlation threshold. Let G_(s) be the union of s and the set of all genes correlated with s. For the instant study, the Spearman coefficient was used as the measure of correlation and 0.7 as the correlation threshold.

3. Compute the correlation coefficient for each pair-wise combination of genes (g_(i),g_(j)) in G_(s). Let X_(s) be the set of all gene pairs for which R(g_(i),g_(j)) is greater than or equal to the correlation threshold. If the genes were plotted as in FIG. 5, there would be an edge (line) between each pair of genes in X_(s).

4. Run the MCE algorithm, as described in Schmidt et al (J. Parallel Distrib. Comput. 69 (2009) 417-428) on the gene pairs X_(s) for each seeding gene.

3.2 Seeded Stacking Algorithm (Steps 5-6)

The purpose of stacking is to reduce the number of cliques down to a manageable number of gene modules (stacks). Continuing with steps 5 and 6 of FIG. 4:

5. For each seeding gene, sort cliques from largest to smallest, i.e. most number of nodes to smallest number of nodes. From the remaining cliques, find the clique with the greatest overlap. If the overlap exceeds a user-specified threshold T, merge the two cliques together to form the first stack. Resort the cliques and stack(s) from largest to smallest and repeat the overlap test and merging. Repeat the process until no new merges occur.

6. One now has a set of stacks for each seeding gene. In the final step, all of the seeded stacks are combined into one set of stacks, a. As the final computation, all of the stacks in a are stacked, just as in step 5. This stack of stacks is the set of gene modules used for the instant study.

Genes that were shown to co-express with genes identified by this method are shown in Tables 8-11. “Stack ID” in the Tables is simply an index to enumerate the stacks and “probeWt” refers to the probe weight, or the number of times a probe (gene) appears in the stack.

TABLE 8 Coexpressed Coexpressed Coexpressed StackID Gene ProbeWt SeedingGene StackID Gene ProbeWt SeedingGene StackID Gene ProbeWt SeedingGene 1 SLCO2B1 1 BGN 1 SPARC 1 SPARC 1 SPARC 1 COL4A1 1 LHFP 1 BGN 1 COL4A1 1 SPARC 1 COL4A1 1 COL4A1 1 ENG 1 BGN 1 COL4A2 1 SPARC 1 HTRA1 1 COL4A1 2 LHFP 1 BGN 2 COL3A1 1 SPARC 2 COL4A1 3 COL4A1 2 THY1 1 BGN 2 SPARC 1 SPARC 2 NID1 3 COL4A1 2 ENG 1 BGN 2 COL4A1 1 SPARC 2 CD93 2 COL4A1 3 COL1A1 1 BGN 2 VCAN 1 SPARC 2 FBN1 2 COL4A1 3 THY1 1 BGN 2 FN1 1 SPARC 2 COL1A1 1 COL4A1 3 ENG 1 BGN 3 HEG1 3 SPARC 2 MCAM 1 COL4A1 4 COL1A1 1 BGN 3 MEF2C 3 SPARC 2 SPARC 1 COL4A1 4 PDGFRB 1 BGN 3 RGS5 2 SPARC 3 COL1A2 4 COL4A1 4 FMNL3 1 BGN 3 KDR 2 SPARC 3 COL4A1 4 COL4A1 5 SLCO2B1 1 BGN 3 LAMA4 1 SPARC 3 VCAN 2 COL4A1 5 LHFP 1 BGN 3 SPARC 1 SPARC 3 FN1 2 COL4A1 5 COL3A1 1 BGN 4 COL3A1 5 SPARC 3 COL1A1 2 COL4A1 6 THY1 1 BGN 4 SPARC 5 SPARC 3 NID1 2 COL4A1 6 LHFP 1 BGN 4 COL1A1 3 SPARC 3 HTRA1 1 COL4A1 6 COL3A1 1 BGN 4 COL1A2 2 SPARC 3 COL6A3 1 COL4A1 7 THY1 1 BGN 4 BGN 2 SPARC 7 COL1A1 1 BGN 4 PDGFRB 2 SPARC 1 INHBA 1 INHBA 7 COL3A1 1 BGN 4 COL4A1 1 SPARC 1 STMN2 1 INHBA 8 BGN 7 BGN 4 IGFBP7 1 SPARC 1 COL10A1 1 INHBA 8 COL1A1 4 BGN 4 FBN1 1 SPARC 8 COL3A1 4 BGN 5 SPARC 4 SPARC 1 THBS2 1 THBS2 8 FMNL3 4 BGN 5 PDGFRB 4 SPARC 1 COL3A1 1 THBS2 8 SLCO2B1 3 BGN 5 DPYSL2 3 SPARC 1 VCAN 1 THBS2 8 SPARC 3 BGN 5 FBN1 3 SPARC 8 ENG 3 BGN 5 HEG1 2 SPARC 8 PDGFRB 3 BGN 5 CDH11 2 SPARC 8 THBS2 1 BGN 5 FBLN5 2 SPARC 1 THBS2 1 COL3A1 5 LAMA2 2 SPARC 1 COL3A1 1 COL3A1 5 IGFBP7 2 SPARC 1 VCAN 1 COL3A1 5 LAMA4 2 SPARC 2 COL3A1 3 COL3A1 5 RGS5 1 SPARC 2 SPARC 3 COL3A1 5 COL4A2 1 SPARC 2 FN1 2 COL3A1 5 COL1A2 1 SPARC 2 COL4A1 2 COL3A1 6 FBN1 7 SPARC 2 VCAN 1 COL3A1 6 LAMA4 6 SPARC 2 COL1A1 1 COL3A1 6 SGK269 5 SPARC 2 FBN1 1 COL3A1 6 CDH11 5 SPARC 3 COL1A2 3 COL3A1 6 DPYSL2 5 SPARC 3 PDGFRB 3 COL3A1 6 LAMA2 5 SPARC 3 IGFBP7 3 COL3A1 6 SPARC 4 SPARC 3 FBN1 3 COL3A1 6 SULF1 4 SPARC 3 CDH11 2 COL3A1 6 FBLN5 3 SPARC 3 AEBP1 2 COL3A1 6 LTBP1 3 SPARC 3 COL3A1 1 COL3A1 6 EPB41L2 3 SPARC 3 SPARC 1 COL3A1 6 MEF2C 3 SPARC 4 COL3A1 5 COL3A1 6 FN1 2 SPARC 4 BGN 4 COL3A1 6 EDIL3 2 SPARC 4 COL1A1 3 COL3A1 6 COL3A1 1 SPARC 4 SPARC 3 COL3A1 6 IGFBP7 1 SPARC 4 FMNL3 2 COL3A1 6 HEG1 1 SPARC 4 PDGFRB 2 COL3A1 4 COL1A2 1 COL3A1 4 THY1 1 COL3A1 4 THBS2 1 COL3A1

TABLE 9 Coexpressed Probe- Seeding Coexpressed Probe- Seeding Coexpressed Seeding StackID Gene Wt Gene StackID Gene Wt Gene StackID Gene ProbeWt Gene 1 DDR2 26870 C7 1 MYH11 168 GSTM2 1 PPAP2B 15794 SRD5A2 1 SPARCL1 25953 C7 1 TGFBR3 163 GSTM2 1 VWA5A 12616 SRD5A2 1 FAT4 24985 C7 1 RBMS3 162 GSTM2 1 SPON1 12395 SRD5A2 1 SYNE1 24825 C7 1 FHL1 161 GSTM2 1 FAT4 12218 SRD5A2 1 SLC8A1 24327 C7 1 MYLK 158 GSTM2 1 SSPN 12126 SRD5A2 1 MEIS1 23197 C7 1 CACHD1 155 GSTM2 1 MKX 11552 SRD5A2 1 PRRX1 22847 C7 1 TIMP3 154 GSTM2 1 PRRX1 11061 SRD5A2 1 CACHD1 22236 C7 1 SYNM 152 GSTM2 1 LOC645954 10811 SRD5A2 1 DPYSL3 20623 C7 1 NEXN 147 GSTM2 1 SYNM 10654 SRD5A2 1 LTBP1 20345 C7 1 MYL9 142 GSTM2 1 ANXA6 10330 SRD5A2 1 SGK269 19461 C7 1 CRYAB 141 GSTM2 1 PDE5A 10011 SRD5A2 1 EDNRA 19280 C7 1 VWA5A 131 GSTM2 1 TSHZ3 9588 SRD5A2 1 TRPC4 18689 C7 1 AOX1 130 GSTM2 1 GSN 9505 SRD5A2 1 TIMP3 18674 C7 1 FLNC 127 GSTM2 1 NID2 9503 SRD5A2 1 TGFBR3 18367 C7 1 PPAP2B 125 GSTM2 1 CLU 9304 SRD5A2 1 ZEB1 18355 C7 1 GSTM2 118 GSTM2 1 TPM2 8659 SRD5A2 1 C1S 16871 C7 1 C21orf63 101 GSTM2 1 FBLN1 8068 SRD5A2 1 ABCC9 16562 C7 1 POPDC2 72 GSTM2 1 PARVA 7949 SRD5A2 1 PCDH18 14936 C7 1 TPM2 66 GSTM2 1 SPOCK3 7772 SRD5A2 1 C7 14789 C7 1 CDC42EP3 60 GSTM2 1 PCDH18 7514 SRD5A2 1 PDGFC 14748 C7 1 CCDC69 58 GSTM2 1 ILK 7078 SRD5A2 1 PTPLAD2 13590 C7 1 CRISPLD2 52 GSTM2 1 ITIH5 6903 SRD5A2 1 VCL 13332 C7 1 GBP2 47 GSTM2 1 ADCY5 6374 SRD5A2 1 MMP2 13107 C7 1 ADCY5 44 GSTM2 1 CRYAB 6219 SRD5A2 1 FERMT2 12681 C7 1 MATN2 40 GSTM2 1 RBMS3 6108 SRD5A2 1 EPB41L2 12335 C7 1 AOC3 38 GSTM2 1 AOX1 4943 SRD5A2 1 PRNP 12133 C7 1 ACACB 36 GSTM2 1 WWTR1 4789 SRD5A2 1 FBN1 11965 C7 1 RND3 28 GSTM2 1 AOC3 4121 SRD5A2 1 GLT8D2 11954 C7 1 CLIP4 26 GSTM2 1 CAP2 4091 SRD5A2 1 DSE 11888 C7 1 APOBEC3C 20 GSTM2 1 MAP1B 3917 SRD5A2 1 SCN7A 11384 C7 1 CAV2 18 GSTM2 1 OGN 3893 SRD5A2 1 PPAP2B 11121 C7 1 TRIP10 17 GSTM2 1 PLN 3581 SRD5A2 1 PGR 10566 C7 1 TCF21 11 GSTM2 1 CFL2 2857 SRD5A2 1 PALLD 10240 C7 1 CAMK2G 11 GSTM2 1 MATN2 2808 SRD5A2 1 CNTN1 10113 C7 1 GSTM5P1 9 GSTM2 1 ADRA1A 2694 SRD5A2 1 SERPING1 9800 C7 1 ACSS3 9 GSTM2 1 BOC 2401 SRD5A2 1 DKK3 9279 C7 1 GSTM4 7 GSTM2 1 ANGPT1 2290 SRD5A2 1 CCND2 9131 C7 1 GSTP1 5 GSTM2 1 POPDC2 2205 SRD5A2 1 MSRB3 8502 C7 1 GSTM1 3 GSTM2 1 FGF2 2162 SRD5A2 1 LAMA4 8477 C7 1 GSTM3 2 GSTM2 1 TCF21 1996 SRD5A2 1 RBMS3 8425 C7 1 GSTM2P1 2 GSTM2 1 LOC283904 1983 SRD5A2 1 FBLN1 7968 C7 1 TGFB3 1 GSTM2 1 DNAJB5 1773 SRD5A2 1 EPHA3 6930 C7 1 FTO 1 IGF1 1 TSPAN2 1731 SRD5A2 1 ACTA2 6824 C7 1 UTP11L 1 IGF1 1 GSTM5 1635 SRD5A2 1 ADAM22 6791 C7 1 SGCB 1 IGF1 1 RGN 1594 SRD5A2 1 WWTR1 6611 C7 2 CHP 14 IGF1 1 PDLIM7 1503 SRD5A2 1 HEPH 6406 C7 2 RP2 14 IGF1 1 MITF 1481 SRD5A2 1 TIMP2 6219 C7 2 SPRYD4 14 IGF1 1 BNC2 1300 SRD5A2 1 CLIC4 6151 C7 2 SGCB 13 IGF1 1 SCN7A 1274 SRD5A2 1 ATP2B4 5897 C7 2 INMT 13 IGF1 1 GPM6B 1202 SRD5A2 1 TNS1 5842 C7 2 IGF1 12 IGF1 1 ARHGAP20 1193 SRD5A2 1 PDGFRA 5802 C7 2 ARPP19 9 IGF1 1 PDZRN4 1190 SRD5A2 1 ITGA1 5781 C7 2 MOCS3 9 IGF1 1 PCP4 1107 SRD5A2 1 RHOJ 5103 C7 2 KATNAL1 8 IGF1 1 ANO5 987 SRD5A2 1 COL14A1 5063 C7 2 C3orf33 8 IGF1 1 C6orf186 930 SRD5A2 1 CALD1 4828 C7 2 SLC16A4 7 IGF1 1 ARHGAP10 793 SRD5A2 1 DCN 4825 C7 2 FTO 7 IGF1 1 CLIP4 775 SRD5A2 1 IRAK3 4476 C7 2 SNX27 6 IGF1 1 CCDC69 733 SRD5A2 1 MATN2 4448 C7 2 C1orf55 5 IGF1 1 SLC24A3 673 SRD5A2 1 KIT 4329 C7 2 C1orf174 4 IGF1 1 ACSS3 668 SRD5A2 1 NEXN 4257 C7 2 SNTN 4 IGF1 1 IL33 611 SRD5A2 1 ZEB2 3798 C7 2 MCART6 4 IGF1 1 CAMK2G 519 SRD5A2 1 COL6A3 3679 C7 2 OTUD3 4 IGF1 1 PTPLA 505 SRD5A2 1 NID2 3678 C7 2 ADAMTS4 4 IGF1 1 EFEMP1 493 SRD5A2 1 PRICKLE2 3671 C7 2 FEZ1 4 IGF1 1 KIT 470 SRD5A2 1 OGN 3418 C7 2 SPATA5 4 IGF1 1 ODZ3 428 SRD5A2 1 SSPN 3142 C7 2 ZNRF3 4 IGF1 1 MRGPRF 390 SRD5A2 1 SORBS1 3126 C7 2 C1orf229 4 IGF1 1 C21orf63 383 SRD5A2 1 PDE5A 2963 C7 2 STX2 4 IGF1 1 CRISPLD2 322 SRD5A2 1 LOC732446 2925 C7 2 PURB 4 IGF1 1 MYADM 314 SRD5A2 1 FCHSD2 2741 C7 2 BVES 4 IGF1 1 C7 278 SRD5A2 1 PMP22 2609 C7 2 DTX3L 4 IGF1 1 PDGFRA 219 SRD5A2 1 TRPC1 2519 C7 2 ZNF713 4 IGF1 1 EYA1 199 SRD5A2 1 ANXA6 2353 C7 2 DSCR3 4 IGF1 1 ATP1A2 174 SRD5A2 1 SPON1 2278 C7 2 SLC35F1 4 IGF1 1 ACACB 173 SRD5A2 1 FBLN5 2115 C7 2 C22orf25 4 IGF1 1 NT5E 168 SRD5A2 1 CHRDL1 1996 C7 2 STK4 4 IGF1 1 GPR124 166 SRD5A2 1 MEF2C 1980 C7 2 EIF5A2 4 IGF1 1 LOC652799 165 SRD5A2 1 EFEMP1 1939 C7 2 SUPT7L 4 IGF1 1 LRCH2 123 SRD5A2 1 JAZF1 1748 C7 2 C10orf78 4 IGF1 1 PYGM 100 SRD5A2 1 DNAJB4 1636 C7 2 ANKS4B 4 IGF1 1 GSTM2 92 SRD5A2 1 ARHGEF6 1594 C7 2 C1orf151 4 IGF1 1 KCNAB1 90 SRD5A2 1 MFAP4 1503 C7 2 RPL32P3 4 IGF1 1 HHIP 82 SRD5A2 1 LOC652799 1470 C7 2 SEC62 4 IGF1 1 ALDH1A2 70 SRD5A2 1 PREX2 1464 C7 2 DBR1 4 IGF1 1 PRDM5 63 SRD5A2 1 MAN1A1 1433 C7 2 FLJ39639 4 IGF1 1 ABCA8 59 SRD5A2 1 TCF21 1224 C7 2 ZNF543 4 IGF1 1 MAML2 51 SRD5A2 1 CRIM1 1181 C7 2 FRRS1 4 IGF1 1 PAK3 38 SRD5A2 1 A2M 1168 C7 2 TATDN3 4 IGF1 1 SNAI2 35 SRD5A2 1 DPYSL2 1029 C7 2 WDR55 4 IGF1 1 UST 27 SRD5A2 1 GPM6B 993 C7 2 KIAA1737 4 IGF1 1 TMLHE 21 SRD5A2 1 PLN 970 C7 2 APOBEC3F 4 IGF1 1 ACTC1 15 SRD5A2 1 IL33 942 C7 2 RNF7 4 IGF1 1 C5orf4 8 SRD5A2 1 CCDC80 889 C7 2 SIKE1 4 IGF1 1 GSTM5P1 4 SRD5A2 1 LMO3 852 C7 2 HSP90B3P 4 IGF1 1 GSTM4 3 SRD5A2 1 SEC23A 765 C7 2 GNS 4 IGF1 1 PDK4 2 SRD5A2 1 MOXD1 708 C7 2 C1orf212 4 IGF1 1 TGFB3 2 SRD5A2 1 SPOCK3 622 C7 2 ZNF70 4 IGF1 1 GSTM1 1 SRD5A2 1 HEG1 608 C7 2 TMEM127 4 IGF1 1 LOC728846 1 TGFB1I1 1 LUM 589 C7 2 ALDH1B1 4 IGF1 1 CLIP3 1 TGFB1I1 1 C7orf58 566 C7 2 HP1BP3 4 IGF1 1 EMILIN1 1 TGFB1I1 1 CDC42EP3 539 C7 2 APOL6 4 IGF1 2 CLIP3 1 TGFB1I1 1 CPVL 524 C7 2 MALL 4 IGF1 2 MRC2 1 TGFB1I1 1 CPA3 421 C7 2 C11orf17 4 IGF1 2 MEG3 1 TGFB1I1 1 SLIT2 417 C7 2 LOC729199 4 IGF1 3 MRC2 1 TGFB1I1 1 KLHL5 376 C7 2 RELL1 4 IGF1 3 LCAT 1 TGFB1I1 1 HLF 322 C7 2 PELI1 4 IGF1 3 MEG3 1 TGFB1I1 1 PLXDC2 313 C7 2 ASB6 4 IGF1 4 LDB3 18 TGFB1I1 1 CAP2 301 C7 2 C2orf18 4 IGF1 4 TGFB1I1 15 TGFB1I1 1 FXYD6 291 C7 2 PSTPIP2 4 IGF1 4 ASB2 11 TGFB1I1 1 ECM2 272 C7 2 CLEC7A 4 IGF1 4 CLIP3 11 TGFB1I1 1 SRD5A2 245 C7 2 RAB22A 4 IGF1 4 ITGA7 10 TGFB1I1 1 MBNL1 245 C7 2 LOC643770 4 IGF1 4 JPH2 10 TGFB1I1 1 LAMA2 169 C7 2 LOC100129502 4 IGF1 4 RUSC2 10 TGFB1I1 1 IL6ST 166 C7 2 ZCCHC4 4 IGF1 4 HRNBP3 8 TGFB1I1 1 PODN 112 C7 2 PNMA2 4 IGF1 4 LIMS2 8 TGFB1I1 1 ATRNL1 110 C7 2 PIGW 4 IGF1 4 CSPG4 7 TGFB1I1 1 DOCK11 60 C7 2 SLC25A32 4 IGF1 4 NLGN3 5 TGFB1I1 1 FGL2 56 C7 2 CLCC1 4 IGF1 4 ADAM33 3 TGFB1I1 1 SPRY2 12 C7 2 KIAA0513 4 IGF1 4 NHSL2 3 TGFB1I1 1 OLFML1 12 C7 2 SS18 4 IGF1 4 SYDE1 2 TGFB1I1 1 NEGR1 4 C7 2 CECR1 4 IGF1 4 RASL12 2 TGFB1I1 1 IGFBP5 1 C7 2 ZNF490 4 IGF1 4 LOC90586 2 TGFB1I1 1 SORBS1 1 DES 2 PDE12 4 IGF1 4 GNAZ 1 TGFB1I1 1 CACNA1C 1 DES 2 C10orf76 4 IGF1 4 TMEM35 1 TGFB1I1 1 DES 1 DES 2 CCL22 4 IGF1 4 LCAT 1 TGFB1I1 2 ITIH5 1 DES 2 RRN3P1 4 IGF1 4 LOC728846 1 TGFB1I1 2 ANXA6 1 DES 2 LOC100127925 4 IGF1 4 SLC24A3 1 TGFB1I1 2 ATP1A2 1 DES 2 SC4MOL 4 IGF1 5 MRGPRF 381 TGFB1I1 3 ITIH5 1 DES 2 AP4E1 4 IGF1 5 PDLIM7 362 TGFB1I1 3 DES 1 DES 2 APOLD1 4 IGF1 5 AOC3 321 TGFB1I1 3 ANXA6 1 DES 2 ARSB 4 IGF1 5 ADCY5 317 TGFB1I1 4 TPM1 1 DES 2 ZNF264 4 IGF1 5 KANK2 306 TGFB1I1 4 DES 1 DES 2 SLC30A6 4 IGF1 5 SLC24A3 292 TGFB1I1 4 CES1 1 DES 2 METTL7A 4 IGF1 5 MYL9 287 TGFB1I1 5 TAGLN 72309 DES 2 PARD6B 4 IGF1 5 FLNC 275 TGFB1I1 5 FLNA 72305 DES 2 STOM 4 IGF1 5 TGFB1I1 253 TGFB1I1 5 TNS1 72049 DES 2 CYP20A1 4 IGF1 5 ITGA7 222 TGFB1I1 5 CNN1 69837 DES 2 LYZ 4 IGF1 5 DES 216 TGFB1I1 5 ACTA2 68389 DES 2 ATP1B4 4 IGF1 5 FLNA 214 TGFB1I1 5 CHRDL1 67725 DES 2 SCD5 4 IGF1 5 EFEMP2 206 TGFB1I1 5 DPYSL3 67225 DES 2 CEP170L 4 IGF1 5 TAGLN 184 TGFB1I1 5 MSRB3 66488 DES 2 NUDT19 4 IGF1 5 RASL12 163 TGFB1I1 5 VCL 65707 DES 2 TXNL4B 4 IGF1 5 GAS6 163 TGFB1I1 5 CCND2 65291 DES 2 APPL1 4 IGF1 5 KCNMB1 163 TGFB1I1 5 SLC8A1 65217 DES 2 OSBPL2 4 IGF1 5 SMTN 157 TGFB1I1 5 MEIS1 65097 DES 2 VMA21 4 IGF1 5 GPR124 140 TGFB1I1 5 ATP2B4 64428 DES 2 NF2 4 IGF1 5 COL6A1 133 TGFB1I1 5 DDR2 64293 DES 2 ZNF772 4 IGF1 5 DNAJB5 127 TGFB1I1 5 LMOD1 64271 DES 2 LOC646973 4 IGF1 5 COL6A2 124 TGFB1I1 5 SORBS1 63359 DES 2 LOC100128096 4 IGF1 5 TPM2 121 TGFB1I1 5 KCNMB1 61499 DES 2 MOAP1 4 IGF1 5 WFDC1 121 TGFB1I1 5 PGR 60803 DES 2 HIGD1A 4 IGF1 5 TNS1 112 TGFB1I1 5 RBPMS 59947 DES 2 DISC2 4 IGF1 5 DKK3 111 TGFB1I1 5 FLNC 59840 DES 2 CYCS 4 IGF1 5 HSPB8 108 TGFB1I1 5 MYLK 58329 DES 2 ZSCAN22 4 IGF1 5 TSPAN18 103 TGFB1I1 5 FHL1 58303 DES 2 LOC646127 4 IGF1 5 MYH11 102 TGFB1I1 5 FZD7 56889 DES 2 RRP15 4 IGF1 5 GEFT 90 TGFB1I1 5 EDNRA 56620 DES 2 LOC100130357 4 IGF1 5 ITIH5 81 TGFB1I1 5 DKK3 56591 DES 2 YES1 4 IGF1 5 PYGM 81 TGFB1I1 5 DES 54990 DES 2 MTFMT 4 IGF1 5 MCAM 78 TGFB1I1 5 PGM5 54713 DES 2 JOSD1 4 IGF1 5 MRVI1 75 TGFB1I1 5 LOC729468 53979 DES 2 RHOF 4 IGF1 5 MYLK 68 TGFB1I1 5 SYNE1 53386 DES 2 LIN54 4 IGF1 5 CNN1 63 TGFB1I1 5 PGM5P2 53378 DES 2 LOC729142 4 IGF1 5 RBPMS2 63 TGFB1I1 5 SPARCL1 52082 DES 2 GNG4 4 IGF1 5 ATP1A2 58 TGFB1I1 5 ACTG2 51556 DES 2 H6PD 4 IGF1 5 LIMS2 58 TGFB1I1 5 TRPC4 51205 DES 2 FBXW2 4 IGF1 5 LMOD1 56 TGFB1I1 5 CAV1 49615 DES 2 NUP43 4 IGF1 5 GNAO1 46 TGFB1I1 5 GNAL 49292 DES 2 WDR5B 4 IGF1 5 LGALS1 43 TGFB1I1 5 TIMP3 48293 DES 2 ANGEL2 4 IGF1 5 DAAM2 41 TGFB1I1 5 ABCC9 46190 DES 2 SGTB 4 IGF1 5 MRC2 39 TGFB1I1 5 MRVI1 44926 DES 2 MAPK1IP1L 4 IGF1 5 HRNBP3 38 TGFB1I1 5 ACTN1 44120 DES 2 ZSCAN29 4 IGF1 5 ASB2 36 TGFB1I1 5 PALLD 43624 DES 2 FXC1 4 IGF1 5 CLIP3 25 TGFB1I1 5 SERPINF1 43602 DES 2 NQO1 4 IGF1 5 C16orf45 22 TGFB1I1 5 JAZF1 42715 DES 2 MOBKL1A 4 IGF1 5 DBNDD2 20 TGFB1I1 5 KANK2 42364 DES 2 ANAPC16 4 IGF1 5 RUSC2 19 TGFB1I1 5 HSPB8 41435 DES 2 C16orf63 4 IGF1 5 RARRES2 18 TGFB1I1 5 MYL9 37460 DES 2 TBCCD1 4 IGF1 5 ADRA1A 18 TGFB1I1 5 PRNP 33800 DES 2 DLEU2 4 IGF1 5 TINAGL1 17 TGFB1I1 5 TSPAN18 33287 DES 2 CARD8 4 IGF1 5 SYNM 17 TGFB1I1 5 FRMD6 32935 DES 2 LOC100130236 4 IGF1 5 TMEM35 14 TGFB1I1 5 CSRP1 32471 DES 2 LOC100130442 4 IGF1 5 COPZ2 12 TGFB1I1 5 HEPH 32337 DES 2 CAMLG 4 IGF1 5 LTBP4 12 TGFB1I1 5 NEXN 29867 DES 2 ZBTB3 4 IGF1 5 SCARA3 11 TGFB1I1 5 PRICKLE2 29746 DES 2 ZNF445 4 IGF1 5 NR2F1 11 TGFB1I1 5 PPAP2B 28983 DES 2 CASP8 4 IGF1 5 PCDH10 11 TGFB1I1 5 MYH11 28923 DES 2 RAB21 4 IGF1 5 RAB34 10 TGFB1I1 5 PDGFC 28732 DES 2 ZC3HAV1L 4 IGF1 5 FOXF1 8 TGFB1I1 5 TPM1 27766 DES 2 SC5DL 4 IGF1 5 TCF7L1 7 TGFB1I1 5 SVIL 27521 DES 2 KILLIN 4 IGF1 5 KIRREL 6 TGFB1I1 5 LOC732446 27335 DES 2 MTX3 4 IGF1 5 DACT1 6 TGFB1I1 5 MEIS2 25944 DES 2 KCNE4 4 IGF1 5 ZNF516 5 TGFB1I1 5 CALD1 25386 DES 2 GM2A 4 IGF1 5 EMILIN1 4 TGFB1I1 5 CNTN1 25377 DES 2 LOC401588 4 IGF1 5 DCHS1 4 TGFB1I1 5 FERMT2 25146 DES 2 C8orf79 4 IGF1 5 EHBP1L1 3 TGFB1I1 5 CLU 24888 DES 2 KIAA0754 4 IGF1 5 SYDE1 2 TGFB1I1 5 SPON1 23171 DES 2 SMU1 4 IGF1 5 PPP1R14A 2 TGFB1I1 5 TGFBR3 23018 DES 2 TSPYL1 4 IGF1 5 SMOC1 2 TGFB1I1 5 CACHD1 22496 DES 2 SPRED1 4 IGF1 5 JPH2 1 TGFB1I1 5 TPM2 22108 DES 2 LOC100128997 4 IGF1 5 MICALL1 1 TGFB1I1 5 GSN 22102 DES 2 LOC729652 4 IGF1 5 LCAT 1 TGFB1I1 5 NID2 21240 DES 2 TRAPPC2 4 IGF1 5 HSPB6 1 TGFB1I1 5 MYOCD 21178 DES 2 KCTD10 4 IGF1 1 FLNA 33418 TPM2 5 MKX 20028 DES 2 DUSP19 4 IGF1 1 TAGLN 33391 TPM2 5 EYA4 19967 DES 2 CCDC122 4 IGF1 1 TNS1 32975 TPM2 5 LOC100127983 18208 DES 2 NXN 4 IGF1 1 CNN1 32489 TPM2 5 ANXA6 16600 DES 2 ZNF283 4 IGF1 1 CHRDL1 31765 TPM2 5 HLF 16262 DES 2 SPATS2L 4 IGF1 1 LMOD1 31568 TPM2 5 VWA5A 16175 DES 2 TRIM5 4 IGF1 1 MYLK 31444 TPM2 5 SRD5A2 16145 DES 2 HAUS3 4 IGF1 1 ACTA2 31310 TPM2 5 SYNM 15943 DES 2 UTP11L 4 IGF1 1 ACTG2 30665 TPM2 5 CDC42EP3 14001 DES 2 SLC30A5 4 IGF1 1 KCNMB1 30331 TPM2 5 AOC3 13787 DES 2 MBOAT1 4 IGF1 1 MSRB3 30007 TPM2 5 TIMP2 13760 DES 2 TERF2 4 IGF1 1 SORBS1 29926 TPM2 5 ILK 13444 DES 2 VPS33A 4 IGF1 1 DPYSL3 29802 TPM2 5 ADCY5 13346 DES 2 SENP5 4 IGF1 1 DES 29158 TPM2 5 PARVA 13266 DES 2 EVI5 4 IGF1 1 VCL 29088 TPM2 5 FBLN1 12617 DES 2 NDUFC2 4 IGF1 1 SLC8A1 29075 TPM2 5 LOC645954 12259 DES 2 ZBTB8A 4 IGF1 1 CCND2 28780 TPM2 5 FAT4 12247 DES 2 ST8SIA4 4 IGF1 1 MEIS1 28764 TPM2 5 ITIH5 11490 DES 2 C7orf64 4 IGF1 1 PGM5 28584 TPM2 5 COL6A3 10595 DES 2 MED18 4 IGF1 1 ATP2B4 28495 TPM2 5 TSHZ3 10118 DES 2 MPV17L 4 IGF1 1 LOC729468 28204 TPM2 5 MCAM 8671 DES 2 C1orf210 4 IGF1 1 FHL1 28101 TPM2 5 MAP1B 8478 DES 2 LIN7C 4 IGF1 1 FLNC 27926 TPM2 5 WFDC1 7000 DES 2 KCNJ11 4 IGF1 1 PGM5P2 27789 TPM2 5 PDE5A 6648 DES 2 COX18 4 IGF1 1 HSPB8 27438 TPM2 5 TLN1 5948 DES 2 PCBD2 4 IGF1 1 DDR2 26679 TPM2 5 PDLIM7 5715 DES 2 SPAST 4 IGF1 1 PGR 26409 TPM2 5 SPOCK3 5657 DES 2 CYP4V2 4 IGF1 1 MRVI1 25979 TPM2 5 BOC 5611 DES 2 LRTOMT 4 IGF1 1 DKK3 25603 TPM2 5 CRYAB 5555 DES 2 IMPAD1 3 IGF1 1 RBPMS 24576 TPM2 5 PMP22 4795 DES 2 UBXN2B 3 IGF1 1 MYH11 24353 TPM2 5 ADRA1A 4611 DES 2 C5orf33 3 IGF1 1 FZD7 24298 TPM2 5 FGF2 4439 DES 2 FOXJ3 3 IGF1 1 TPM2 23458 TPM2 5 CELF2 4392 DES 2 PPP1R15B 3 IGF1 1 GNAL 23091 TPM2 5 MMP2 4243 DES 2 GNAI3 2 IGF1 1 MYL9 22987 TPM2 5 WWTR1 3966 DES 2 SAR1B 2 IGF1 1 JAZF1 21665 TPM2 5 CAP2 3592 DES 2 SERPINB9 2 IGF1 1 CAV1 21569 TPM2 5 LOC100129846 3236 DES 2 PTGIS 2 IGF1 1 KANK2 21564 TPM2 5 RBMS3 3165 DES 2 C3orf70 2 IGF1 1 EDNRA 20876 TPM2 5 AOX1 3042 DES 2 RUNDC2B 2 IGF1 1 SPARCL1 20468 TPM2 5 MFAP4 3011 DES 2 SYT11 1 IGF1 1 TRPC4 19698 TPM2 5 TCF21 2881 DES 1 CPXM2 1 ITGA7 1 TSPAN18 18763 TPM2 5 MATN2 2851 DES 1 MRVI1 1 ITGA7 1 ACTN1 18284 TPM2 5 MRGPRF 2724 DES 1 ITGA7 1 ITGA7 1 TIMP3 18017 TPM2 5 POPDC2 2704 DES 2 ADCY5 661 ITGA7 1 ABCC9 17793 TPM2 5 CFL2 2404 DES 2 MRGPRF 652 ITGA7 1 SYNE1 17659 TPM2 5 LOC283904 2374 DES 2 PDLIM7 649 ITGA7 1 SERPINF1 17306 TPM2 5 PRELP 2253 DES 2 FLNC 627 ITGA7 1 PALLD 16659 TPM2 5 CCDC69 2088 DES 2 KANK2 624 ITGA7 1 PRICKLE2 16570 TPM2 5 PLN 2046 DES 2 MYL9 611 ITGA7 1 CSRP1 15853 TPM2 5 DNAJB5 1956 DES 2 AOC3 602 ITGA7 1 HEPH 14646 TPM2 5 GPR124 1851 DES 2 FLNA 540 ITGA7 1 NEXN 13548 TPM2 5 GAS6 1830 DES 2 TAGLN 527 ITGA7 1 MYOCD 13479 TPM2 5 TSPAN2 1830 DES 2 KCNMB1 492 ITGA7 1 MEIS2 13043 TPM2 5 ANGPT1 1797 DES 2 DES 491 ITGA7 1 TPM1 12988 TPM2 5 MFGE8 1766 DES 2 ITGA7 481 ITGA7 1 SPON1 12334 TPM2 5 ITGA1 1682 DES 2 SLC24A3 434 ITGA7 1 EYA4 12112 TPM2 5 GSTM5 1596 DES 2 TNS1 423 ITGA7 1 HLF 11972 TPM2 5 MYADM 1579 DES 2 TSPAN18 364 ITGA7 1 SYNM 11833 TPM2 5 CES1 1511 DES 2 MCAM 351 ITGA7 1 SVIL 11249 TPM2 5 CAMK2G 1453 DES 2 TPM2 322 ITGA7 1 FRMD6 10974 TPM2 5 PCP4 1361 DES 2 MYLK 322 ITGA7 1 CNTN1 10796 TPM2 5 SLC24A3 1275 DES 2 HSPB8 317 ITGA7 1 CLU 10687 TPM2 5 RGN 1215 DES 2 MYH11 317 ITGA7 1 LOC100127983 10582 TPM2 5 KCNMA1 1050 DES 2 MRVI1 314 ITGA7 1 PRNP 10088 TPM2 5 PDZRN4 876 DES 2 LMOD1 301 ITGA7 1 MKX 9903 TPM2 5 ARHGAP10 867 DES 2 CNN1 288 ITGA7 1 CALD1 9712 TPM2 5 C6orf186 841 DES 2 ITIH5 287 ITGA7 1 FERMT2 9315 TPM2 5 ARHGAP20 828 DES 2 DNAJB5 282 ITGA7 1 NID2 9290 TPM2 5 FXYD6 826 DES 2 CHRDL1 264 ITGA7 1 ITIH5 8936 TPM2 5 PTGER2 802 DES 2 EFEMP2 256 ITGA7 1 PDGFC 8919 TPM2 5 SLC12A4 721 DES 2 ATP1A2 239 ITGA7 1 LOC732446 8793 TPM2 5 NID1 670 DES 2 SMTN 238 ITGA7 1 LOC645954 8764 TPM2 5 ITGA9 568 DES 2 GAS6 231 ITGA7 1 ADCY5 8698 TPM2 5 SMTN 558 DES 2 WFDC1 222 ITGA7 1 AOC3 8557 TPM2 5 TCEAL2 557 DES 2 TGFB1I1 220 ITGA7 1 SRD5A2 8415 TPM2 5 COL6A1 499 DES 2 GPR124 206 ITGA7 1 GSN 7427 TPM2 5 ITGA5 475 DES 2 NID2 204 ITGA7 1 WFDC1 6345 TPM2 5 ATP1A2 417 DES 2 ADRA1A 197 ITGA7 1 VWA5A 6297 TPM2 5 C21orf63 408 DES 2 PYGM 189 ITGA7 1 ILK 6243 TPM2 5 EFEMP2 389 DES 2 RASL12 186 ITGA7 1 TGFBR3 5718 TPM2 5 PTPLA 366 DES 2 BOC 184 ITGA7 1 CDC42EP3 5544 TPM2 5 ST5 364 DES 2 FZD7 174 ITGA7 1 TSHZ3 5478 TPM2 5 JAM3 350 DES 2 ACTG2 172 ITGA7 1 FAT4 4923 TPM2 5 ITGA7 333 DES 2 PRICKLE2 157 ITGA7 1 PARVA 4922 TPM2 5 LPP 320 DES 2 GEFT 156 ITGA7 1 MCAM 4880 TPM2 5 COL6A2 302 DES 2 COL6A1 142 ITGA7 1 PDLIM7 4753 TPM2 5 ODZ3 294 DES 2 PGM5 133 ITGA7 1 ADRA1A 4540 TPM2 5 PLEKHO1 266 DES 2 SYNM 132 ITGA7 1 ANXA6 4499 TPM2 5 PYGM 249 DES 2 FHL1 126 ITGA7 1 FBLN1 4133 TPM2 5 TINAGL1 239 DES 2 HEPH 112 ITGA7 1 BOC 3515 TPM2 5 PCDH10 238 DES 2 COL6A2 110 ITGA7 1 COL6A3 3490 TPM2 5 PNMA1 232 DES 2 LOC729468 109 ITGA7 1 CRYAB 3436 TPM2 5 ACACB 221 DES 2 MYOCD 101 ITGA7 1 SPOCK3 3141 TPM2 5 RASL12 213 DES 2 ACTA2 66 ITGA7 1 PDE5A 2530 TPM2 5 LARGE 182 DES 2 RBPMS2 62 ITGA7 1 MAP1B 2406 TPM2 5 GEFT 181 DES 2 LIMS2 53 ITGA7 1 FGF2 2375 TPM2 5 NCS1 176 DES 2 GNAO1 45 ITGA7 1 LOC100129846 2231 TPM2 5 TRANK1 173 DES 2 ASB2 44 ITGA7 1 MRGPRF 2029 TPM2 5 FGFR1 166 DES 2 HRNBP3 43 ITGA7 1 DNAJB5 2029 TPM2 5 AHNAK2 164 DES 2 POPDC2 41 ITGA7 1 LOC283904 2007 TPM2 5 LGALS1 156 DES 2 DAAM2 38 ITGA7 1 POPDC2 1965 TPM2 5 RRAS 133 DES 2 ODZ3 34 ITGA7 1 TCF21 1785 TPM2 5 C2orf40 132 DES 2 PDZRN4 33 ITGA7 1 TLN1 1720 TPM2 5 TGFB1I1 126 DES 2 C6orf186 30 ITGA7 1 CELF2 1700 TPM2 5 RAB34 95 DES 2 ITGA9 28 ITGA7 1 AOX1 1459 TPM2 5 PTRF 94 DES 2 NID1 27 ITGA7 1 SLC24A3 1296 TPM2 5 SCHIP1 91 DES 2 C16orf45 22 ITGA7 1 CCDC69 1287 TPM2 5 GSTM2 87 DES 2 RUSC2 22 ITGA7 1 ANGPT1 1256 TPM2 5 MAOB 49 DES 2 TMEM35 19 ITGA7 1 PCP4 1226 TPM2 5 MASP1 48 DES 2 CLIP3 19 ITGA7 1 BNC2 1170 TPM2 5 TRIP10 45 DES 2 MRC2 19 ITGA7 1 PDZRN4 1069 TPM2 5 RARRES2 40 DES 2 TINAGL1 17 ITGA7 1 RGN 1065 TPM2 5 RBPMS2 37 DES 2 DBNDD2 17 ITGA7 1 CES1 1060 TPM2 5 APOBEC3C 30 DES 2 ITGB3 11 ITGA7 1 GPR124 917 TPM2 5 COPZ2 29 DES 2 LDB3 9 ITGA7 1 GAS6 888 TPM2 5 CACNA1C 21 DES 2 ITGA5 9 ITGA7 1 CFL2 871 TPM2 5 GNAO1 16 DES 2 NCS1 9 ITGA7 1 CAMK2G 869 TPM2 5 UST 12 DES 2 FOXF1 8 ITGA7 1 ARHGAP20 850 TPM2 5 ACTC1 12 DES 2 DACT1 7 ITGA7 1 GSTM5 794 TPM2 5 CES4 11 DES 2 CSPG4 6 ITGA7 1 CAP2 752 TPM2 5 ID4 11 DES 2 JPH2 6 ITGA7 1 PRELP 693 TPM2 5 C16orf45 10 DES 2 ZNF516 6 ITGA7 1 SMTN 540 TPM2 5 LIMS2 9 DES 2 KIRREL 3 ITGA7 1 FXYD6 533 TPM2 5 GSTM5P1 6 DES 2 NHSL2 3 ITGA7 1 TSPAN2 500 TPM2 5 GSTM4 5 DES 2 LCAT 2 ITGA7 1 KCNMA1 488 TPM2 5 CBX7 3 DES 2 FABP3 2 ITGA7 1 PTGER2 429 TPM2 5 PPP1R14A 3 DES 2 GNAZ 1 ITGA7 1 TCEAL2 425 TPM2 5 FABP3 3 DES 2 P2RX1 1 ITGA7 1 MYADM 402 TPM2 5 GSTM1 2 DES 1 SLC8A1 47139 SRD5A2 1 JAM3 360 TPM2 5 GSTM2P1 2 DES 1 LOC729468 47056 SRD5A2 1 COL6A1 354 TPM2 5 HSPB6 1 DES 1 DPYSL3 47002 SRD5A2 1 ATP1A2 339 TPM2 1 GSTM5P1 4 GSTM1 1 ACTA2 46967 SRD5A2 1 SLC12A4 327 TPM2 1 GSTM2 4 GSTM1 1 PGM5 46874 SRD5A2 1 ITGA5 325 TPM2 1 GSTM4 4 GSTM1 1 MEIS1 46871 SRD5A2 1 ITGA9 301 TPM2 1 GSTM5 4 GSTM1 1 ACTG2 46703 SRD5A2 1 ITGA7 300 TPM2 1 SPOCK3 3 GSTM1 1 PGM5P2 46699 SRD5A2 1 EFEMP2 298 TPM2 1 PGM5 3 GSTM1 1 MSRB3 46428 SRD5A2 1 PYGM 254 TPM2 1 HSPB8 3 GSTM1 1 TAGLN 46404 SRD5A2 1 COL6A2 248 TPM2 1 AOX1 2 GSTM1 1 FLNA 46365 SRD5A2 1 ARHGAP10 227 TPM2 1 CSRP1 2 GSTM1 1 VCL 46278 SRD5A2 1 PNMA1 211 TPM2 1 FLNC 2 GSTM1 1 CNN1 45892 SRD5A2 1 RASL12 207 TPM2 1 DES 2 GSTM1 1 CHRDL1 45879 SRD5A2 1 GEFT 194 TPM2 1 GSTM2P1 2 GSTM1 1 TNS1 45774 SRD5A2 1 PTPLA 183 TPM2 1 GSTM1 2 GSTM1 1 ATP2B4 45519 SRD5A2 1 CRISPLD2 181 TPM2 1 CAV1 1 GSTM1 1 LMOD1 44299 SRD5A2 1 ACSS3 177 TPM2 1 SRD5A2 1 GSTM1 1 PGR 44126 SRD5A2 1 AHNAK2 175 TPM2 1 GSTM3 1 GSTM1 1 SORBS1 43839 SRD5A2 1 ST5 175 TPM2 1 LOC729468 1 GSTM1 1 CCND2 43401 SRD5A2 1 PLEKHO1 165 TPM2 1 EYA4 1 GSTM1 1 DDR2 43389 SRD5A2 1 LARGE 164 TPM2 1 PGM5P2 1 GSTM1 1 EDNRA 42947 SRD5A2 1 C21orf63 151 TPM2 1 CSRP1 364 GSTM2 1 FHL1 41382 SRD5A2 1 TINAGL1 150 TPM2 1 CAV1 358 GSTM2 1 KCNMB1 41204 SRD5A2 1 ACACB 138 TPM2 1 TNS1 358 GSTM2 1 TRPC4 40884 SRD5A2 1 LGALS1 136 TPM2 1 ATP2B4 356 GSTM2 1 SYNE1 40118 SRD5A2 1 TGFB1I1 126 TPM2 1 MEIS2 352 GSTM2 1 CAV1 39836 SRD5A2 1 ITGB3 123 TPM2 1 FLNA 350 GSTM2 1 SPARCL1 39359 SRD5A2 1 RRAS 123 TPM2 1 TAGLN 350 GSTM2 1 RBPMS 38414 SRD5A2 1 NCS1 107 TPM2 1 GNAL 350 GSTM2 1 FZD7 34246 SRD5A2 1 PTRF 94 TPM2 1 DPYSL3 348 GSTM2 1 SRD5A2 33968 SRD5A2 1 LPP 91 TPM2 1 MEIS1 347 GSTM2 1 DKK3 33963 SRD5A2 1 C2orf40 90 TPM2 1 TRPC4 345 GSTM2 1 JAZF1 33635 SRD5A2 1 MAOB 59 TPM2 1 CCND2 325 GSTM2 1 MYLK 33158 SRD5A2 1 GSTM2 51 TPM2 1 SYNE1 321 GSTM2 1 ABCC9 33072 SRD5A2 1 TRIP10 48 TPM2 1 EDNRA 317 GSTM2 1 GNAL 32392 SRD5A2 1 ALDH1A2 43 TPM2 1 ACTA2 313 GSTM2 1 PALLD 31713 SRD5A2 1 RARRES2 36 TPM2 1 PALLD 310 GSTM2 1 FLNC 29309 SRD5A2 1 COPZ2 34 TPM2 1 FRMD6 309 GSTM2 1 PRICKLE2 29168 SRD5A2 1 APOBEC3C 34 TPM2 1 PGM5 301 GSTM2 1 MRVI1 28467 SRD5A2 1 RBPMS2 33 TPM2 1 HSPB8 293 GSTM2 1 TIMP3 28313 SRD5A2 1 DBNDD2 31 TPM2 1 ACTG2 287 GSTM2 1 FRMD6 28108 SRD5A2 1 GNAO1 19 TPM2 1 CNN1 286 GSTM2 1 PRNP 28106 SRD5A2 1 ACTC1 15 TPM2 1 PGM5P2 285 GSTM2 1 HSPB8 26756 SRD5A2 1 CES4 11 TPM2 1 SLC8A1 282 GSTM2 1 PDGFC 26571 SRD5A2 1 C16orf45 10 TPM2 1 LOC729468 276 GSTM2 1 CNTN1 26148 SRD5A2 1 GSTP1 8 TPM2 1 PRICKLE2 275 GSTM2 1 EYA4 26129 SRD5A2 1 UST 5 TPM2 1 SRD5A2 275 GSTM2 1 MEIS2 25616 SRD5A2 1 GSTM4 4 TPM2 1 RBPMS 275 GSTM2 1 MYOCD 25477 SRD5A2 1 GSTM5P1 4 TPM2 1 PDGFC 270 GSTM2 1 NEXN 25068 SRD5A2 1 CBX7 3 TPM2 1 EYA4 270 GSTM2 1 CACHD1 25049 SRD5A2 1 PPP1R14A 3 TPM2 1 MYOCD 262 GSTM2 1 FERMT2 24208 SRD5A2 1 FABP3 3 TPM2 1 CALD1 255 GSTM2 1 LOC100127983 23635 SRD5A2 1 C15orf51 1 TPM2 1 KCNMB1 250 GSTM2 1 TPM1 22943 SRD5A2 1 GSTM2P1 1 TPM2 1 ACTN1 227 GSTM2 1 CALD1 22765 SRD5A2 1 FZD7 216 GSTM2 1 SERPINF1 22186 SRD5A2 1 LOC100127983 216 GSTM2 1 CSRP1 21728 SRD5A2 1 DKK3 209 GSTM2 1 ACTN1 21590 SRD5A2 1 GSTM5 207 GSTM2 1 HLF 21402 SRD5A2 1 CHRDL1 204 GSTM2 1 DES 20952 SRD5A2 1 SORBS1 202 GSTM2 1 MYL9 19970 SRD5A2 1 SPOCK3 202 GSTM2 1 HEPH 19688 SRD5A2 1 JAZF1 189 GSTM2 1 TSPAN18 19099 SRD5A2 1 LMOD1 180 GSTM2 1 SVIL 18819 SRD5A2 1 DES 172 GSTM2 1 TGFBR3 18423 SRD5A2 1 MYH11 18066 SRD5A2 1 KANK2 17638 SRD5A2 1 CDC42EP3 16173 SRD5A2

TABLE 10 StackID Coexpressed Gene ProbeWt Seeding Gene 1 NCAPH 9758 CDC20 1 CDC20 9758 CDC20 1 IQGAP3 9758 CDC20 1 ESPL1 9674 CDC20 1 CENPA 9671 CDC20 1 POC1A 9671 CDC20 1 KIF18B 9328 CDC20 1 WDR62 9316 CDC20 1 TROAP 9178 CDC20 1 ADAMTS7 8987 CDC20 1 PKMYT1 8875 CDC20 1 SLC2A6 8875 CDC20 1 FNDC1 8554 CDC20 1 FAM64A 8346 CDC20 1 FAM131B 8322 CDC20 1 PNLDC1 8135 CDC20 1 KIFC1 7598 CDC20 1 C9orf100 7547 CDC20 1 RPS6KL1 7527 CDC20 1 MRAP 7521 CDC20 1 AURKB 7224 CDC20 1 C2orf54 7150 CDC20 1 TMEM163 6853 CDC20 1 KRBA1 6846 CDC20 1 ZMYND10 6825 CDC20 1 LOC541473 6824 CDC20 1 SLC6A1 6669 CDC20 1 DQX1 6601 CDC20 1 BAI2 6583 CDC20 1 EME1 6533 CDC20 1 CICP3 6481 CDC20 1 PPFIA4 6480 CDC20 1 PADI1 6458 CDC20 1 SSPO 6431 CDC20 1 GABRB2 6422 CDC20 1 IRF5 6399 CDC20 1 NXPH1 6399 CDC20 1 ZIC1 6346 CDC20 1 SLC6A20 6336 CDC20 1 PKD1L1 6281 CDC20 1 BIRC5 6278 CDC20 1 AQP10 6251 CDC20 1 ABCA4 6216 CDC20 1 TFR2 6181 CDC20 1 LOC646070 6179 CDC20 1 CSPG5 6165 CDC20 1 CENPM 6124 CDC20 1 EFNA3 6100 CDC20 1 GPC2 6078 CDC20 1 HYAL3 6047 CDC20 1 CELA3B 6031 CDC20 1 LOC100287112 6015 CDC20 1 SRCRB4D 5999 CDC20 1 DNAJB3 5993 CDC20 1 PADI3 5989 CDC20 1 PAX8 5971 CDC20 1 AIM1L 5971 CDC20 1 FAM131C 5915 CDC20 1 PRRT4 5915 CDC20 1 MLXIPL 5915 CDC20 1 E2F1 5912 CDC20 1 E2F7 5893 CDC20 1 RAD54L 5888 CDC20 1 C1orf81 5813 CDC20 1 NFKBIL2 5742 CDC20 1 LOC729061 5728 CDC20 1 TAS1R3 5671 CDC20 1 VWA3B 5643 CDC20 1 MYBL2 5565 CDC20 1 TTLL6 5531 CDC20 1 LOC100130097 5525 CDC20 1 CHRNG 5491 CDC20 1 TTBK1 5491 CDC20 1 TRIM46 5491 CDC20 1 MST1R 5491 CDC20 1 EXOC3L 5474 CDC20 1 TH 5474 CDC20 1 CHST1 5474 CDC20 1 LOC442676 5439 CDC20 1 CNTN2 5435 CDC20 1 DPYSL5 5435 CDC20 1 C3orf20 5368 CDC20 1 NPC1L1 5291 CDC20 1 CICP5 5281 CDC20 1 KLRG2 5275 CDC20 1 CCDC108 5275 CDC20 1 IL28B 5217 CDC20 1 CELSR3 5166 CDC20 1 RNFT2 5138 CDC20 1 C17orf53 5114 CDC20 1 TRPC2 5095 CDC20 1 KCNA1 5078 CDC20 1 C8G 4946 CDC20 1 COL11A1 4685 CDC20 1 C1orf222 4673 CDC20 1 SLC6A12 4633 CDC20 1 HCN3 4608 CDC20 1 GTSE1 4528 CDC20 1 ORC1L 4497 CDC20 1 STX1A 4475 CDC20 1 MFSD2A 4451 CDC20 1 BEST4 4389 CDC20 1 CACNA1E 4299 CDC20 1 KLHDC7A 4297 CDC20 1 MAPK15 4272 CDC20 1 GHRHR 4211 CDC20 1 KEL 4155 CDC20 1 C2orf62 4113 CDC20 1 ANXA9 4063 CDC20 1 RAET1G 4059 CDC20 1 GPR88 3913 CDC20 1 F12 3749 CDC20 1 LYPD1 3681 CDC20 1 C2orf70 3665 CDC20 1 ABCB9 3638 CDC20 1 MSLNL 3589 CDC20 1 CDC25C 3573 CDC20 1 CELA3A 3551 CDC20 1 AQP12B 3551 CDC20 1 NEU4 3551 CDC20 1 KIF2C 3541 CDC20 1 NEIL3 3426 CDC20 1 NUDT17 3399 CDC20 1 ULBP2 3395 CDC20 1 KIF17 3341 CDC20 1 ARHGEF19 3340 CDC20 1 CYP4A22 3317 CDC20 1 CYP4A11 3317 CDC20 1 SCNN1D 3311 CDC20 1 FRMD1 3219 CDC20 1 FAM179A 3194 CDC20 1 NDUFA4L2 3109 CDC20 1 LCE2D 2984 CDC20 1 ODZ4 2936 CDC20 1 ABCC12 2809 CDC20 1 DPF1 2750 CDC20 1 CDH24 2653 CDC20 1 LOC154449 2641 CDC20 1 KIF21B 2534 CDC20 1 SEMA5B 2499 CDC20 1 PSORS1C2 2497 CDC20 1 FCRL4 2434 CDC20 1 FUT6 2313 CDC20 1 TRAIP 2258 CDC20 1 E2F8 2232 CDC20 1 SLC38A3 2199 CDC20 1 CBX2 2174 CDC20 1 CDCA5 2130 CDC20 1 DUSP5P 2080 CDC20 1 GPAT2 1997 CDC20 1 AVPR1B 1991 CDC20 1 MGC50722 1990 CDC20 1 AQP12A 1983 CDC20 1 C6orf222 1965 CDC20 1 PRAMEF19 1965 CDC20 1 PRAMEF18 1965 CDC20 1 SLC5A9 1965 CDC20 1 FCN3 1955 CDC20 1 GCM2 1910 CDC20 1 ADORA3 1862 CDC20 1 PLA2G2F 1821 CDC20 1 C6orf25 1765 CDC20 1 CDC45 1681 CDC20 1 AGXT 1529 CDC20 1 KIF25 1507 CDC20 1 ZDHHC19 1507 CDC20 1 APLNR 1374 CDC20 1 TACC3 1220 CDC20 1 TK1 1063 CDC20 1 C15orf42 1052 CDC20 1 FANCA 990 CDC20 1 GINS4 932 CDC20 1 MCM10 757 CDC20 1 CYB561D1 748 CDC20 1 FUT5 687 CDC20 1 POLQ 632 CDC20 1 LOC643988 621 CDC20 1 RAD51 601 CDC20 1 DGAT2 582 CDC20 1 KIF24 566 CDC20 1 CDCA3 442 CDC20 1 CLSPN 381 CDC20 1 ESYT3 356 CDC20 1 EXO1 278 CDC20 1 CDCA2 186 CDC20 1 CKAP2L 159 CDC20 1 FOXM1 157 CDC20 1 FEN1 136 CDC20 1 UHRF1 125 CDC20 1 KIF20A 110 CDC20 1 ESCO2 107 CDC20 1 CA2 100 CDC20 1 PLK1 84 CDC20 1 PTTG1 64 CDC20 1 KIF14 53 CDC20 1 CIT 42 CDC20 1 FAM54A 39 CDC20 1 CDCA8 28 CDC20 1 DEPDC1B 12 CDC20 1 MYBL2 12208 MYBL2 1 BIRC5 12208 MYBL2 1 TROAP 12208 MYBL2 1 ESPL1 12190 MYBL2 1 WDR62 12032 MYBL2 1 KIF18B 12015 MYBL2 1 FAM64A 11915 MYBL2 1 PKMYT1 11774 MYBL2 1 SLC2A6 11774 MYBL2 1 GTSE1 11356 MYBL2 1 E2F1 11062 MYBL2 1 AURKB 11010 MYBL2 1 RNFT2 10720 MYBL2 1 CENPM 10651 MYBL2 1 CENPA 10628 MYBL2 1 POC1A 10289 MYBL2 1 FDXR 10285 MYBL2 1 NFKBIL2 10214 MYBL2 1 E2F7 10195 MYBL2 1 C9orf100 10103 MYBL2 1 CDH24 10094 MYBL2 1 ABCB9 10079 MYBL2 1 NDUFA4L2 9961 MYBL2 1 ADAMTS7 9614 MYBL2 1 MAST1 9313 MYBL2 1 GABBR2 9262 MYBL2 1 MYH7B 8759 MYBL2 1 DNAH3 8637 MYBL2 1 TTLL6 8619 MYBL2 1 ZFHX2 8592 MYBL2 1 CDC20 8589 MYBL2 1 RASAL1 8452 MYBL2 1 NCAPH 8273 MYBL2 1 IQGAP3 8245 MYBL2 1 DNAH2 8219 MYBL2 1 LOC400499 8151 MYBL2 1 CHST1 8037 MYBL2 1 ATP4A 7868 MYBL2 1 TH 7731 MYBL2 1 EXOC3L 7603 MYBL2 1 E2F8 7590 MYBL2 1 MMP11 7465 MYBL2 1 CELP 7344 MYBL2 1 CDCA5 7024 MYBL2 1 FAM131B 6981 MYBL2 1 C14orf73 6896 MYBL2 1 FBXW9 6802 MYBL2 1 PLEKHG6 6725 MYBL2 1 FNDC1 6720 MYBL2 1 SEZ6 6515 MYBL2 1 FCHO1 6413 MYBL2 1 APLNR 6402 MYBL2 1 ALAS2 6382 MYBL2 1 VSX1 6360 MYBL2 1 LOC197350 6312 MYBL2 1 DPF1 6205 MYBL2 1 CDC45 6026 MYBL2 1 C11orf9 6020 MYBL2 1 EME1 6010 MYBL2 1 ADAMTS13 5896 MYBL2 1 TMEM145 5896 MYBL2 1 C8G 5840 MYBL2 1 CBX2 5838 MYBL2 1 TMEM210 5659 MYBL2 1 CCDC135 5593 MYBL2 1 ADAMTS14 5571 MYBL2 1 ITGA2B 5337 MYBL2 1 POLD1 5286 MYBL2 1 PNLDC1 5146 MYBL2 1 UCP3 5123 MYBL2 1 FANCA 5068 MYBL2 1 MSLNL 5061 MYBL2 1 TEPP 4930 MYBL2 1 LRRC16B 4901 MYBL2 1 CACNA1F 4901 MYBL2 1 EFNB3 4887 MYBL2 1 MYBPC2 4851 MYBL2 1 FUT6 4847 MYBL2 1 CDH15 4847 MYBL2 1 HAL 4809 MYBL2 1 PGA3 4720 MYBL2 1 PGA4 4720 MYBL2 1 C17orf53 4717 MYBL2 1 UMODL1 4713 MYBL2 1 OTOG 4690 MYBL2 1 DBH 4661 MYBL2 1 POM121L9P 4629 MYBL2 1 DNAJB13 4394 MYBL2 1 TK1 4360 MYBL2 1 C9orf117 4336 MYBL2 1 RHBDL1 4308 MYBL2 1 MUC5B 4283 MYBL2 1 SPAG4 4276 MYBL2 1 GOLGA7B 4111 MYBL2 1 APOB48R 4107 MYBL2 1 IQCD 3984 MYBL2 1 FUT5 3977 MYBL2 1 AIFM3 3973 MYBL2 1 LOC390595 3868 MYBL2 1 CYP27B1 3833 MYBL2 1 SUSD2 3824 MYBL2 1 TGM6 3767 MYBL2 1 CDCA3 3765 MYBL2 1 C20orf151 3706 MYBL2 1 C11orf41 3650 MYBL2 1 C9orf98 3636 MYBL2 1 KRT24 3589 MYBL2 1 ABCC12 3582 MYBL2 1 B3GNT4 3569 MYBL2 1 AZI1 3556 MYBL2 1 RLTPR 3427 MYBL2 1 KIF24 3264 MYBL2 1 DERL3 3232 MYBL2 1 LIPE 3221 MYBL2 1 TTLL9 3196 MYBL2 1 SEC1 3196 MYBL2 1 ADAM8 3185 MYBL2 1 SLC25A19 3136 MYBL2 1 PRSS27 3136 MYBL2 1 ODF3L2 3094 MYBL2 1 ODZ4 3034 MYBL2 1 RAD54L 2936 MYBL2 1 KCNE1L 2936 MYBL2 1 SBF1P1 2915 MYBL2 1 AIPL1 2868 MYBL2 1 UNC13A 2862 MYBL2 1 REM2 2832 MYBL2 1 KIFC1 2808 MYBL2 1 TSNAXIP1 2799 MYBL2 1 LOC390660 2767 MYBL2 1 SLC6A12 2762 MYBL2 1 WDR16 2723 MYBL2 1 ACR 2710 MYBL2 1 TMPRSS13 2672 MYBL2 1 C15orf42 2659 MYBL2 1 DNMT3B 2649 MYBL2 1 UNC13D 2610 MYBL2 1 SYT5 2544 MYBL2 1 PAX2 2462 MYBL2 1 PRCD 2426 MYBL2 1 PPFIA3 2421 MYBL2 1 GCGR 2338 MYBL2 1 CACNG3 2289 MYBL2 1 LAIR2 2233 MYBL2 1 MCM10 2178 MYBL2 1 C2orf54 2172 MYBL2 1 LOC400419 2138 MYBL2 1 RINL 2136 MYBL2 1 DKFZp451A211 2118 MYBL2 1 LAMA1 2060 MYBL2 1 C9orf169 2060 MYBL2 1 CATSPER1 2001 MYBL2 1 OPCML 1896 MYBL2 1 C9orf50 1852 MYBL2 1 DOC2GP 1760 MYBL2 1 TACC3 1665 MYBL2 1 APOBEC3A 1632 MYBL2 1 LOC728307 1606 MYBL2 1 PDIA2 1572 MYBL2 1 LTB4R2 1419 MYBL2 1 OIP5 1393 MYBL2 1 ORC1L 1340 MYBL2 1 GSG2 1268 MYBL2 1 FSD1 1256 MYBL2 1 CDC25C 1228 MYBL2 1 KSR2 1183 MYBL2 1 DGAT2 1183 MYBL2 1 KIF2C 1180 MYBL2 1 RAD51 1178 MYBL2 1 FNDC8 1178 MYBL2 1 RAB3IL1 991 MYBL2 1 UHRF1 936 MYBL2 1 ENO4 855 MYBL2 1 C10orf105 780 MYBL2 1 NEIL3 733 MYBL2 1 PPBP 672 MYBL2 1 PROCA1 671 MYBL2 1 TMEM132A 647 MYBL2 1 DHRS2 548 MYBL2 1 PLK1 523 MYBL2 1 GINS4 485 MYBL2 1 CEL 480 MYBL2 1 ZNF367 406 MYBL2 1 FOXM1 402 MYBL2 1 POLQ 319 MYBL2 1 ADAM12 312 MYBL2 1 SEMA7A 284 MYBL2 1 HOXB5 137 MYBL2 1 EXO1 115 MYBL2 1 KIF4A 114 MYBL2 1 FEN1 112 MYBL2 1 CLSPN 107 MYBL2 1 CIT 94 MYBL2 1 CDCA2 85 MYBL2 1 KIF4B 68 MYBL2 1 PIK3R5 56 MYBL2 1 KIF20A 52 MYBL2 1 ZWINT 31 MYBL2 1 SPAG5 19 MYBL2 1 ERCC6L 17 MYBL2 1 TPX2 11 TPX2 1 TOP2A 11 TPX2 1 NUSAP1 10 TPX2 1 MELK 7 TPX2 1 RACGAP1 6 TPX2 1 NCAPG 4 TPX2 1 MKI67 4 TPX2 1 CDKN3 4 TPX2 1 PRC1 4 TPX2 1 ARHGAP11B 3 TPX2 1 KIAA0101 3 TPX2 1 ANLN 3 TPX2 1 FAM111B 2 TPX2 1 RRM2 1 TPX2 1 KIF11 1 TPX2 1 PRR11 1 TPX2 1 CENPF 1 TPX2 2 MKI67 41 TPX2 2 CASC5 39 TPX2 2 ASPM 38 TPX2 2 KIF4A 36 TPX2 2 DLGAP5 36 TPX2 2 KIF4B 36 TPX2 2 TPX2 33 TPX2 2 KIF14 31 TPX2 2 EXO1 31 TPX2 2 SKA3 30 TPX2 2 SPAG5 27 TPX2 2 CIT 27 TPX2 2 BUB1 26 TPX2 2 CDKN3 26 TPX2 2 CENPF 25 TPX2 2 MELK 20 TPX2 2 ANLN 19 TPX2 2 BUB1B 18 TPX2 2 UBE2C 17 TPX2 2 CEP55 16 TPX2 2 KIF20A 15 TPX2 2 DEPDC1B 15 TPX2 2 DTL 14 TPX2 2 UBE2T 13 TPX2 2 NCAPG 13 TPX2 2 PBK 13 TPX2 2 DIAPH3 10 TPX2 2 KIF23 6 TPX2 2 FOXM1 5 TPX2 2 RRM2 3 TPX2 2 SGOL1 2 TPX2 2 PLK1 2 TPX2 2 CCNA2 2 TPX2 2 CDK1 2 TPX2 2 NUSAP1 1 TPX2

TABLE 11 StackID Coexpressed Gene ProbeWt Seeding Gene 1 NNT 1 DUSP1 1 RNF180 1 DUSP1 1 PCDH18 1 DUSP1 2 RNF180 1 DUSP1 2 DUSP1 1 DUSP1 2 PCDH18 1 DUSP1 3 ACTB 1 DUSP1 3 RHOB 1 DUSP1 3 DUSP1 1 DUSP1 4 ACTB 1 DUSP1 4 DUSP1 1 DUSP1 4 CRTAP 1 DUSP1 5 RNF180 1 DUSP1 5 DUSP1 1 DUSP1 5 CRTAP 1 DUSP1 5 PAM 1 DUSP1 6 DUSP1 8 DUSP1 6 NR4A1 7 DUSP1 6 FOS 7 DUSP1 6 EGR1 5 DUSP1 6 BTG2 5 DUSP1 6 FOSB 5 DUSP1 6 JUN 4 DUSP1 6 NR4A2 3 DUSP1 6 TIPARP 3 DUSP1 6 CYR61 3 DUSP1 6 ATF3 2 DUSP1 6 RHOB 2 DUSP1 6 NEDD9 2 DUSP1 6 MCL1 1 DUSP1 6 RASD1 1 DUSP1 1 JUNB 1 EGR1 1 TIPARP 1 EGR1 1 BTG2 1 EGR1 2 JUNB 1 EGR1 2 BTG2 1 EGR1 2 EGR1 1 EGR1 3 KLF4 1 EGR1 3 FOSB 1 EGR1 3 EGR1 1 EGR1 4 FOSB 1 EGR1 4 CSRNP1 1 EGR1 4 EGR1 1 EGR1 5 EGR1 35 EGR1 5 FOS 30 EGR1 5 NR4A1 25 EGR1 5 FOSB 23 EGR1 5 BTG2 22 EGR1 5 CYR61 20 EGR1 5 ZFP36 18 EGR1 5 CSRNP1 17 EGR1 5 NR4A3 13 EGR1 5 EGR3 13 EGR1 5 KLF6 12 EGR1 5 RHOB 11 EGR1 5 DUSP1 10 EGR1 5 ATF3 9 EGR1 5 JUN 9 EGR1 5 TIPARP 8 EGR1 5 NFKBIZ 7 EGR1 5 NR4A2 7 EGR1 5 JUNB 7 EGR1 5 IER2 7 EGR1 5 MCL1 4 EGR1 5 KLF4 4 EGR1 5 EGR2 4 EGR1 5 NEDD9 2 EGR1 5 SRF 2 EGR1 5 GADD45B 1 EGR1 5 TRIB1 1 EGR1 1 FOS 14 FOS 1 BTG2 14 FOS 1 CSRNP1 13 FOS 1 ZFP36 13 FOS 1 JUNB 9 FOS 1 NR4A3 7 FOS 1 FOSB 7 FOS 1 SIK1 6 FOS 1 BHLHE40 6 FOS 1 RHOB 5 FOS 1 TIPARP 5 FOS 1 KLF6 5 FOS 1 MCL1 5 FOS 1 NR4A1 4 FOS 1 EGR1 4 FOS 1 NR4A2 4 FOS 1 GADD45B 3 FOS 1 SOCS3 2 FOS 1 NFKBIZ 1 FOS 2 FOS 24 FOS 2 FOSB 22 FOS 2 EGR1 20 FOS 2 NR4A1 19 FOS 2 BTG2 18 FOS 2 ZFP36 12 FOS 2 CSRNP1 11 FOS 2 CYR61 10 FOS 2 DUSP1 8 FOS 2 ATF3 8 FOS 2 IER2 7 FOS 2 RHOB 6 FOS 2 TIPARP 6 FOS 2 NR4A2 6 FOS 2 JUN 6 FOS 2 JUNB 6 FOS 2 EGR3 5 FOS 2 NR4A3 4 FOS 2 KLF6 3 FOS 2 PPP1R15A 2 FOS 2 NEDD9 2 FOS 2 KLF4 2 FOS 2 EGR2 2 FOS 2 MCL1 1 FOS 1 EMP1 7 GADD45B 1 BHLHE40 7 GADD45B 1 SOCS3 7 GADD45B 1 NR4A3 4 GADD45B 1 FOSL2 3 GADD45B 1 GADD45B 3 GADD45B 1 RNF122 3 GADD45B 1 KLF10 3 GADD45B 1 CSRNP1 3 GADD45B 1 SLC2A3 2 GADD45B 1 ZFP36 1 GADD45B 2 FOSB 2 GADD45B 2 NR4A1 2 GADD45B 2 FOS 2 GADD45B 2 GADD45B 2 GADD45B 2 BTG2 2 GADD45B 2 NR4A3 2 GADD45B 2 JUNB 2 GADD45B 2 EGR1 2 GADD45B 2 CSRNP1 2 GADD45B 2 ZFP36 2 GADD45B 2 RHOB 1 GADD45B 2 EGR3 1 GADD45B 2 ATF3 1 GADD45B 3 GADD45B 4 GADD45B 3 JUNB 4 GADD45B 3 CSRNP1 4 GADD45B 3 ZFP36 4 GADD45B 3 SOCS3 4 GADD45B 3 RHOB 3 GADD45B 3 BHLHE40 3 GADD45B 3 FOS 2 GADD45B 3 FOSL2 2 GADD45B 3 BTG2 2 GADD45B 3 NR4A3 2 GADD45B 3 FOSB 1 GADD45B 3 IRF1 1 GADD45B 1 FOSL2 1 ZFP36 1 HBEGF 1 ZFP36 1 BHLHE40 1 ZFP36 2 HBEGF 1 ZFP36 2 NR4A3 1 ZFP36 2 BHLHE40 1 ZFP36 3 CSRNP1 53 ZFP36 3 ZFP36 49 ZFP36 3 JUNB 29 ZFP36 3 FOS 26 ZFP36 3 BHLHE40 24 ZFP36 3 BTG2 24 ZFP36 3 FOSB 20 ZFP36 3 NR4A3 18 ZFP36 3 SOCS3 18 ZFP36 3 EGR1 16 ZFP36 3 RHOB 16 ZFP36 3 FOSL2 15 ZFP36 3 NR4A1 15 ZFP36 3 GADD45B 10 ZFP36 3 MYADM 9 ZFP36 3 KLF6 8 ZFP36 3 CYR61 8 ZFP36 3 EGR3 8 ZFP36 3 EMP1 8 ZFP36 3 LMNA 7 ZFP36 3 TIPARP 7 ZFP36 3 NR4A2 7 ZFP36 3 MCL1 6 ZFP36 3 SIK1 6 ZFP36 3 ATF3 6 ZFP36 3 CEBPD 5 ZFP36 3 IER3 5 ZFP36 3 IER2 5 ZFP36 3 MAFF 4 ZFP36 3 IRF1 4 ZFP36 3 RNF122 4 ZFP36 3 SRF 3 ZFP36 3 ERRFI1 2 ZFP36 3 SLC25A25 2 ZFP36 3 CDKN1A 2 ZFP36 3 EGR2 2 ZFP36 3 KLF4 1 ZFP36

Example 4: Prospective Validation Study of RS27

Study Design and Statistical Methods

The algorithm RS27 in Table 4 was tested in a prospective clinical validation study that included 395 evaluable patients who had surgery for their prostate cancer between 1997 and 2010 at the University of California, San Francisco (UCSF). The patients had Low or Intermediate risk (by CAPRA) for clinically localized prostate cancer who might have been reasonable candidates for active surveillance but underwent RP at UCSF within 6 months of the diagnosis of prostate cancer by biopsy. No randomization for patient selection was performed. For each patient, prostate biopsy samples from one fixed, paraffin-embedded tissue (FPET) block containing one or more tumor-containing needle cores was evaluated.

To investigate if there is a significant relationship between RS27 or any component of RS27 and adverse pathology at RP, multivariable and univariable multinomial logistic regression models were used and p-values from likelihood-ratio (LR) tests of the null hypothesis that the odds ratio (OR) is one were reported. The multinomial logistic model was also used to calculate estimates with 95% confidence intervals of the probability of high-grade or non-organ confined disease. To evaluate the relationship between RS27, baseline covariates, and combinations of these factors with high grade or non-organ confined disease, multivariable and univariable binary logistic regression models were used and p-values from likelihood-ratio tests of the null hypothesis that the odds ratio (OR) is one were reported.

The primary endpoint was formulated as follows:

TABLE 12 Clinical Endpoint-RP Grade and Stage RP Gleason Score Pathologic T2 Stage Pathologic T3 Stage ≤3 + 3 1 2    3 + 4 3 4 Major pattern 4 or 5 6 minor pattern 5, or tertiary pattern 5 where Gleason Score≤3+3 and pT2 (denoted “1”) is the reference category and all other categories (2-6) are compared to the reference category.

Cell combinations of Table 12 evaluated in binary logistic regression models include the following:

-   -   Cells 2, 4, 6 vs. 1, 3, 5: Non-organ-confined disease     -   Cells 5, 6 vs. 1, 2, 3, 4: High-grade disease     -   Cells 2, 4, 5, 6 vs. 1 and 3: High-grade or non-organ-confined         disease         RS27 Algorithm

RS27 on a scale from 0 to 100 was derived from reference-normalized gene expression measurements as follows.

Unscaled RS27 (RS27u) was defined as in Table 4: RS27u=0.735*ECM (Stromal Response) group−0.368*Migration (Cellular Organization) group−0.352*PSA (Androgen) group+0.095*Proliferation (TPX2)

Where: ECM (Stromal Response) group score=0.527*BGN+0.457*COL1A1+0.156*SFRP4 Migration (Cellular Organization) group score=0.163*FLNC+0.504*GSN+0.421*TPM2+0.394*GSTM2 PSA (Androgen) group score=0.634*FAM13C+1.079*KLK2+0.642*AZGP1+0.997*SRD5A2 Thresh Proliferation (TPX2) score=TPX2 Thresh

where the thresholded gene scores for SRD5A2 and TPX2 are calculated as follows:

${{SRD5A}\; 2\mspace{14mu}{Thresh}} = \left\{ {{\begin{matrix} 5.5 & {{{if}\mspace{14mu}{SRD5A}\; 2} < 5.5} \\ {SRD5A2} & {otherwise} \end{matrix}{TPX}\; 2\mspace{14mu}{Thresh}} = \left\{ \begin{matrix} 5.0 & {{{if}\mspace{14mu}{TPX}\; 2}\  < 5.0} \\ {TPX2} & {otherwise} \end{matrix} \right.} \right.$

RS27u is then rescaled to be between 0 and 100 as follows:

${RS}\; 27({scaled})\left\{ \begin{matrix} 0 & {{{if}\mspace{14mu} 13.4 \times \left( {{RS27u} + {10.5}} \right)} < 0} \\ {13.4 \times \left( {{{RS}\; 27u} + {10{.5}}} \right)} & {{{if}\mspace{14mu} 0} \leq {13.4 \times \left( {{{RS}\; 27u} + 10.5} \right)} \leq 100} \\ 100 & {{{if}\mspace{14mu} 13.4 \times \left( {{RS27u} + {10{.5}}} \right)} > 100} \end{matrix} \right.$

Patients were classified into low, intermediate, and high RS27 groups using pre-specified cut-points defined in Table 13 below. These cut-points defined the boundaries between low and intermediate RS27 groups and between intermediate and high RS27 groups. The cutpoints were derived from the discovery study with the intent of identifying substantial proportions of patients who on average had clinically meaningful low or high risk of adverse pathology. The RS27 was rounded to the nearest integer before the cut-points defining the RS27 groups were applied.

TABLE 13 RS27 Group Score Low Less than 16 Intermediate Greater than or equal to 16 and less than 30 High Greater than or equal to 30 Assay Methods

Paraffin from the samples was removed with Shandon Xylene substitute (Thermo Scientific, Kalamazoo, Mich.). Nucleic acids were isolated using the Agencourt® FormaPure® XP kit (Beckman Coulter, Beverly, Mass.).

The amount of RNA was determined using the Quant-iT™ RiboGreen® RNA Assay kit (Invitrogen™, Carlsbad, Calif.). Quantitated RNA was convereted to complementary DNA using the Omniscript® RT kit (Qiagen, Valencia, Calif.) and combined with the reverse primers for the 12 genes of RS27 and 5 normalization genes (ARF1, ATP5E, CLTC, GPS1, PGK1) as shown in Table A. The reaction was incubated at 37° C. for 60 minutes and then inactivated at 93° C. for 5 minutes.

The cDNA was preamplified using a custom TaqMan® PreAmp Master Mix made for Genomic Health, Inc. by Life Technologies (Carlsbad, Calif.) and the forward and reverse primers for all targets as shown in Table A. The reaction was placed in a thermocycler (DNA Engine® PTC 200G, Bio-Rad, Hercules, Calif.) and incubated under the following conditions: A) 95° C. for 15 sec; B) 60° C. for 4 min; C) 95° C. for 15 sec; and D) steps B and C were repeated 8 times. The amplified product was then mixed with the forward and reverse primers and probes for each of the targets as shown in Table A and the QuantiTect® Primer Assay master mix (Qiagen, Valencia, Calif.) and amplified for 45 cycles in a LightCycler® 480 (Roche Applied Science, Indianapolis, Ind.). The level of expression was calculated using the crossing-point (Cp) method.

Results

RS27 significantly predicted for adverse pathology, non-organ-confined disease, high-grade disease, and high-grade or non-organ-confined disease, and adds value beyond biopsy Gleason Score as shown in Tables 14, 15, 16, and 17, respectively.

TABLE 14 Prediction of Adverse Pathology Variable LR Chi-Square DF P-value RS27 Score 19.31 5 0.002 Central Biopsy Gleason Score 32.86 5 <0.001 3 + 4 vs 3 + 3 Results obtained from the multivariable multinomial logistic model for cells 2, 3, 4, 5, and 6 vs 1 in Table 12. DF = degrees of freedom

TABLE 15 Prediction of Non-organ-confined disease 95% Odds Confidence LR Chi- P- Model Variables Ratio Interval DF Square value Uni- RS27 2.20 (1.46, 3.31) 1 14.44 <0.001 variable Model Multi- RS27 1.93 (1.25, 2.96) 1 8.97 0.003 variable Central Biopsy 1.79 (1.04, 3.10) 1 4.23 0.040 Model Gleason Score 3 + 4 vs 3 + 3 Results obtained from univariable and multivariable binary logistic regression models for cells 2, 4, 6 vs. 1, 3, 5 in Table 12 Odds ratio for RS27 was per 20 unit increase

TABLE 16 Prediction of high-grade disease 95% Odds Confidence LR Chi- P- Model Variables Ratio Interval DF Square value Uni- RS27 2.48 (1.60, 3.85) 1 16.78 <0.001 variable Model Multi- RS27 2.32 (1.46, 3.67) 1 12.92 <0.001 variable Central Biopsy 1.36 (0.75, 2.47) 1 0.98 0.322 Model Gleason Score 3 + 4 vs 3 + 3 Results obtained from univariable and multivariable binary logistic regression models for cells 5, 6 vs. 1-4 in Table 12 Odds ratio for RS27 was per 20 unit increase

TABLE 17 Prediction of high-grade or non-organ-confined disease 95% Odds Confidence LR Chi- P- Model Variables Ratio Interval DF Square value Uni- RS27 2.23 (1.52, 3.27) 1 17.77 <0.001 variable Model Multi- RS27 1.93 (1.30, 2.88) 1 10.70 0.001 variable Central Biopsy 1.94 (1.17, 3.21) 1 6.45 0.011 Model Gleason Score 3 + 4 vs 3 + 3 *Results obtained from univariable and multivariable binary logistic regression models for cells 2, 4, 5, 6 vs. 1, 3 in Table 12 Odds ratio for RS17 was per 20 unit increase

In addition, RS27 predicted adverse pathology beyond conventional clinical/pathology treatment factors as shown in Table 18.

TABLE 18 Prediction of Adverse Pathology Beyond Conventional Clinical/Pathology Treatment Factors Variable LR Chi-Square DF P-value RS27 21.46 5 <0.001 Original Biopsy Gleason Score 22.77 5 <0.001 RS27 19.31 5 0.002 Central Biopsy Gleason Score 32.86 5 <0.001 RS27 30.09 5 <0.001 Clin T2 v. T1 11.94 5 0.036 RS27 30.17 5 <0.001 Baseline PSA (ng/ml) <10 v. >= 10 10.44 5 0.064 RS27 30.75 5 <0.001 Continuous PSA 15.17 5 0.010 RS27 26.36 5 <0.001 Age 19.05 5 0.002 RS27 29.20 5 <0.001 Pct Core Positive 4.75 5 0.447

When added to conventional clinical/pathology tools such as CAPRA, RS27 further refined the risk of high grade or non-organ-confined disease. Using CAPRA alone, 5% of patients were identified as having greater than 85% probability of being free from high-grade or non-organ-confined disease compared to 22% of patients identified of being free from high-grade or non-organ-confined disease using RS27 in addition to CAPRA (FIG. 8).

When added to conventional clinical/pathology tools such as AUA (D'Amico et al., JAMA 280:969-974, 1998), RS27 further refined the risk of high grade or non-organ-confined disease. As shown in FIG. 9, using AUA alone, 0% of patients are identified as having greater than 80% probability of being free from high-grade or non-organ-confined disease compared to 27% of patients identified of being free from high-grade or non-organ-confined disease using GPS in addition to AUA.

Individual genes and gene groups of RS27 were also associated with adverse pathology, high-grade disease, non-organ-confined disease, and high-grade or non-organ-confined disease, in univariable analyses as shown in Tables 19, 20, 21, and 22, respectively.

TABLE 19 Association of Genes and Gene Groups with Adverse Pathology, Univariable Analyses Genes and Gene Groups LR Chi-Square DF P-value BGN 7.11 5 0.213 COL1A1 7.88 5 0.163 SFRP4 8.87 5 0.114 FLNC 12.26 5 0.031 GSN 5.73 5 0.333 GSTM2 1.84 5 0.870 TPM2 18.33 5 0.003 AZGP1 22.87 5 <0.001 KLK2 5.97 5 0.309 FAM13C1 21.55 5 <0.001 SRD5A2 9.10 5 0.105 SRD5A2 Thresholded 9.25 5 0.099 TPX2 14.26 5 0.014 TPX2 Thresholded 23.34 5 <0.001 Ref Gene Average 3.27 5 0.659 Stromal Group Score 9.84 5 0.080 Cellular Organization Group 8.04 5 0.154 Score Androgen Group Score 29.46 5 <0.001 Proliferation Group Score 23.34 5 <0.001 GPS 29.98 5 <0.001

TABLE 20 Association of Genes and Gene Groups with High-Grade Disease, Univariable Analyses Gene Chi-Square DF P-value OR (95% CI) BGN 3.67 1 0.055 1.46 (0.99, 2.15) COL1A1 2.33 1 0.127 1.32 (0.93, 1.87) SFRP4 6.08 1 0.014 1.33 (1.06, 1.67) FLNC 3.04 1 0.081 0.77 (0.57, 1.03) GSN 0.14 1 0.710 0.94 (0.67, 1.32) GSTM2 0.03 1 0.870 0.97 (0.69, 1.37) TPM2 2.85 1 0.091 0.76 (0.56, 1.04) AZGP1 12.69 1 <0.001 0.58 (0.42, 0.79) KLK2 3.50 1 0.061 0.62 (0.38, 1.02) FAM13C1 9.29 1 0.002 0.51 (0.33, 0.79) SRD5A2 3.26 1 0.071 0.76 (0.56, 1.02) SRD5A2 Thresholded 2.70 1 0.100 0.75 (0.53, 1.06) TPX2 1.72 1 0.190 1.21 (0.91, 1.59) TPX2 Thresholded 7.38 1 0.007 1.93 (1.20, 3.11) Ref Gene Average 1.18 1 0.277 0.86 (0.65, 1.14) Stromal Response Group Score 4.92 1 0.027 1.49 (1.05, 2.12) Cellular Organization Group 1.12 1 0.290 0.87 (0.66, 1.13) Score Androgen Group Score 15.07 1 <0.001 0.69 (0.58, 0.83) Proliferation Group Score 7.38 1 0.007 1.93 (1.20, 3.11)

TABLE 21 Association of Genes and Gene Groups with Non-Organ-Confined Disease, Univariable Analyses Gene Chi-Square DF p-value Odds Ratio 95% CI BGN 2.58 1 0.109 1.34 (0.94, 1.91) COL1A1 2.90 1 0.089 1.33 (0.96, 1.83) SFRP4 4.39 1 0.036 1.25 (1.01, 1.54) FLNC 0.34 1 0.560 0.92 (0.70, 1.21) GSN 0.27 1 0.603 0.92 (0.67, 1.26) GSTM2 0.16 1 0.693 0.94 (0.68, 1.29) TPM2 0.51 1 0.473 0.9 (0.67, 1.20) AZGP1 12.48 1 <0.001 0.59 (0.44, 0.80) KLK2 2.10 1 0.148 0.71 (0.45, 1.12) FAM13C1 12.42 1 0.000 0.48 (0.32, 0.73) SRD5A2 2.42 1 0.120 0.8 (0.60, 1.06) SRD5A2 Thresholded 2.65 1 0.103 0.77 (0.56, 1.06) TPX2 6.38 1 0.012 1.39 (1.08, 1.81) TPX2 Thresholded 6.51 1 0.011 1.82 (1.14, 2.89) Ref Gene Average 0.33 1 0.563 0.93 (0.73, 1.19) Stromal Response Group Score 4.24 1 0.040 1.41 (1.02, 1.95) Cellular Organization Group Score 0.45 1 0.504 0.92 (0.72, 1.18) Androgen Group Score 14.64 1 <0.001 0.71 (0.60, 0.85) Proliferation Group Score 6.51 1 0.011 1.82 (1.14, 2.89)

TABLE 22 Association of Genes and Gene Groups with High-Grade or Non-Organ-Confined Disease, Univariable Analyses Gene Chi-Square DF p-value Odds Ratio 95% CI BGN 3.15 1 0.076 1.33 (0.97, 1.84) COL1A1 1.96 1 0.162 1.23 (0.92, 1.65) SFRP4 7.08 1 0.008 1.29 (1.07, 1.55) FLNC 2.36 1 0.125 0.83 (0.65, 1.06) GSN 0.45 1 0.503 0.91 (0.68, 1.20) GSTM2 0.49 1 0.484 0.9 (0.68, 1.20) TPM2 2.24 1 0.135 0.82 (0.63, 1.06) AZGP1 12.20 1 0.001 0.61 (0.46, 0.82) KLK2 2.18 1 0.140 0.73 (0.48, 1.11) FAM13C1 11.13 1 0.001 0.53 (0.37, 0.78) SRD5A2 4.36 1 0.037 0.76 (0.59, 0.98) SRD5A2 Thresholded 4.63 1 0.032 0.73 (0.55, 0.98) TPX2 3.50 1 0.062 1.25 (0.99, 1.58) TPX2 Thresholded 5.86 1 0.016 1.73 (1.09, 2.74) Ref Gene Average 0.68 1 0.041 0.91 (0.73, 1.14) Stromal Response Group Score 4.59 1 0.032 1.37 (1.03, 1.84) Cellular Organization Group 1.54 1 0.215 0.87 (0.70, 1.08) Score Androgen Group Score 16.56 1 <0.001 0.72 (0.61, 0.84) Proliferation Group Score 5.86 1 0.016 1.73 (1.09, 2.74)

Example 5: RS27 Adds Value Beyond PTEN/TMPRSS2-ERG Status in Predicting Clinical Recurrence

PTEN mutation and TMPRSS2-ERG fusion genes are commonly associated with poor prognosis in prostate cancer. Here, RS27 was analyzed to determine whether it can provide value beyond PTEN/TMPRSS2-ERG status in predicting clinical recurrence.

PTEN and TMPRSS2-ERG fusion expression levels obtained in the gene identification study described in Example 1 above and in U.S. Pub. No. 20120028264 were used to stratify patients into PTEN low and PTEN normal groups. PTEN and TMPRSS2-ERG (“T2-ERG”) status of the patients were found as follows:

TABLE 23 Distribution of PTEN Expression by T2-ERG Status T2-ERG Negative (53%) T2-ERG Positive (47%) Median PTEN 8.9 8.7 25% PTEN 8.7 8.4

A cutpoint for “PTEN low” was made at <=8.5, which included approximately 13% of T2-ERG negative patients and 28% of T2-ERG positive patients. PTEN normal was defined as >8.5.

Univaraible Cox Proportional Hazards was applied to evaluate the association between PTEN status and time to clinical recurrence (cR). FIG. 10 and Table 24 show that PTEN low patients have a higher risk of recurrence compared to PTEN normal patients.

TABLE 24 Chi Sq P-value HR 95% CI 12.44 <0.001 0.38 (0.22, 0.65)

When the patients were further stratified into PTEN low/T2-ERG negative (“category 0”), PTEN low/T2-ERG positive (“category 1”), PTEN normal/T2-ERG negative (“category 2”), and PTEN normal/T2-ERG positive (“category 3”), both PTEN low categories had the lowest recurrence rates compared to PTEN normal patients as shown in FIG. 11 and Table 25.

TABLE 25 PTEN/T2-ERG categories CHISQ P-VALUE 95% CI Cat 1 v 0 0.93 0.34 (0.28, 1.55) Cat 2 v 0 11.80 <0.01 (0.12, 0.56) Cat 3 v 0 7.05 0.01 (0.16, 0.76)

The tables below summarize the results of a multivariable model with PTEN/T2-ERG status (Table 26) or PTEN status (Table 27), RS27, and biopsy Gleason Score (Bx GS), demonstrating that RS27 adds value beyond PTEN and T2-ERG markers and Biopsy GS in predicting clinical recurrence.

TABLE 26 VARIABLE DF CHISQ P-VALUE HR 95% CI RS27 1 64.13 <0.01 1.07 (1.05, 1.09) PTEN/T2-ERG Status 3 1.59 0.66 PTEN/T2-ERG 1 0.06 0.80 0.91 (0.41, 1.98) (Cat 1 v. 0) PTEN/T2-ERG 1 1.17 0.28 0.65 (0.29, 1.42) (Cat 2 v. 0) PTEN/T2-ERG 1 0.14 0.71 0.86 (0.39, 1.89) (Cat 3 v. 0) BX GS 2 7.19 0.03 Bx GS (7 v. 6) 1 6.86 0.01 0.40 (0.20, 0.79) Bx GS (8+ v. 6) 1 1.35 0.24 0.69 (0.36, 1.29)

TABLE 27 VARIABLE DF CHISQ P-VALUE HR 95% CI GPS 1 66.67 <0.01 1.07 (1.05, 1.09) PTEN Status 1 0.86 0.35 0.78 (0.46, 1.32) BX GS 2 6.43 0.04 Bx GS (7 v. 6) 1 6.12 0.01 0.42 (0.21, 0.84) Bx GS (8+ v. 6) 1 1.15 0.28 0.71 (0.37, 1.33)

TABLE A Official Symbol: Sequence ID: SEQ ID NO: Forward Primer Sequence: SEQ ID NO: Reverse Primer Sequence: SEQ ID NO: Probe Sequence: ALDH1A2 NM_170696.1   1 CACGTCTGTCCCTCTCTGCT   2 GACCGTGGCTCAACTTTGTAT   3 TCTCTGTAGGGCCCAGCTCTCAGG ANPEP NM_001150.2   5 CCACCTTGGACCAAAGTAAAGC   6 TCTCAGCGTCACCTGGTAGGA   7 CTCCCCAACACGCTGAAACCCG AR NM_000044.2   9 CGACTTCACCGCACCTGAT  10 TGACACAAGTGGGACTGGGATA  11 ACCATGCCGCCAGGGTACCACA ARF1 NM_001658.2  13 CAGTAGAGATCCCCGCAACT  14 ACAAGCACATGGCTATGGAA  15 CTTGTCCTTGGGTCACCCTGCA ASPN NM_017680.4  17 CATTGCCACTTCAACTCTAA  18 ATTGTTAGTGTCCAGGCTCT  19 TATCCCTTTGGAAGACCTTGCTTG ATP5E NM_006886.2  21 CCGCTTTCGCTACAGCAT  22 TGGGAGTATCGGATGTAGCTG  23 TCCAGCCTGTCTCCAGTAGGCCAC AZGP1 NM_001185.2  25 GAGGCCAGCTAGGAAGCAA  26 CAGGAAGGGCAGCTACTGG  27 TCTGAGATCCCACATTGCCTCCAA BGN NM_001711.3  29 GAGCTCCGCAAGGATGAC  30 CTTGTTGTTCACCAGGACGA  31 CAAGGGTCTCCAGCACCTCTACGC BIN1 NM_004305.1  33 CCTGCAAAAGGGAACAAGAG  34 CGTGGTTGACTCTGATCTCG  35 CTTCGCCTCCAGATGGCTCCC BMP6 NM_001718.4  37 GTGCAGACCTTGGTTCACCT  38 CTTAGTTGGCGCACAGCAC  39 TGAACCCCGAGTATGTCCCCAAAC C7 NM_000587.2  41 ATGTCTGAGTGTGAGGCGG  42 AGGCCTTATGCTGGTGACAG  43 ATGCTCTGCCCTCTGCATCTCAGA CADM1 NM_014333.2  45 CCACCACCATCCTTACCATC  46 GATCCACTGCCCTGATCG  47 TCTTCACCTGCTCGGGAATCTGTG CD276 NM_001024736.1  49 CCAAAGGATGCGATACACAG  50 GGATGACTTGGGAATCATGTC  51 CCACTGTGCAGCCTTATTTCTCCAATG CD44 NM_000610.3  53 GGCACCACTGCTTATGAAGG  54 GATGCTCATGGTGAATGAGG  55 ACTGGAACCCAGAAGCACACCCTC CDC20 NM_001255.2  57 AGTGACCTGCACTCGCTGCT  58 GGCTTCCTTGGCTTTGCGCT  59 CCAATGCACCCCCTGCGCGCTGGC CDKN2C NM_001262.2  61 TGAAGGGAACCTGCCCTTGCA  62 TGTGCTTCACCAGGAACTCCACC  63 TGGCTGCCAAAGAAGGCCACCTCCGGGT CLTC NM_004859.1  65 ACCGTATGGACAGCCACAG  66 TGACTACAGGATCAGCGCTTC  67 TCTCACATGCTGTACCCAAAGCCA COL1A1 NM_000088.2  69 GTGGCCATCCAGCTGACC  70 CAGTGGTAGGTGATGTTCTGGGA  71 TCCTGCGCCTGATGTCCACCG COL1A2 NM_000089.2  73 CAGCCAAGAACTGGTATAGGAGCT  74 AAACTGGCTGCCAGCATTG  75 TCTCCTAGCCAGACGTGTTTCTTGTCCTTG COL3A1 NM_000090.3  77 GGAGGTTCTGGACCTGCTG  78 ACCAGGACTGCCACGTTC  79 CTCCTGGTCCCCAAGGTGTCAAAG COL4A1 NM_001845.4  81 ACAAAGGCCTCCCAGGAT  82 GAGTCCCAGGAAGACCTGCT  83 CTCCTTTGACACCAGGGATGCCAT COL5A2 NM_000393.3  85 GGTCGAGGAACCCAAGGT  86 GCCTGGAGGTCCAACTCTG  87 CCAGGAAATCCTGTAGCACCAGGC COL6A1 NM_001848.2  89 GGAGACCCTGGTGAAGCTG  90 TCTCCAGGGACACCAACG  91 CTTCTCTTCCCTGATCACCCTGCG COL8A1 NM_001850.3  93 TGGTGTTCCAGGGCTTCT  94 CCCTGTAAACCCTGATCCC  95 CCTAAGGGAGAGCCAGGAATCCCA CSF1 NM_000757.3  97 TGCAGCGGCTGATTGACA  98 CAACTGTTCCTGGTCTACAAACTCA  99 TCAGATGGAGACCTCGTGCCAAATTACA CSRP1 NM_004078.1 101 ACCCAAGACCCTGCCTCT 102 GCAGGGGTGGAGTGATGT 103 CCACCCTTCTCCAGGGACCCTTAG CYP3A5 NM_000777.2 105 TCATTGCCCAGTATGGAGATG 106 GACAGGCTTGCCTTTCTCTG 107 TCCCGCCTCAAGTTTCTCACCAAT DES NM_001927.3 109 ACTTCTCACTGGCCGACG 110 GCTCCACCTTCTCGTTGGT 111 TGAACCAGGAGTTTCTGACCACGC DPP4 NM_001935.3 113 GTCCTGGGATCGGGAAGT 114 GTACTCCCACCGGGATACAG 115 CGGCTATTCCACACTTGAACACGC DUSP1 NM_004417.2 117 AGACATCAGCTCCTGGTTCA 118 GACAAACACCCTTCCTCCAG 119 CGAGGCCATTGACTTCATAGACTCCA EGR1 NM_001964.2 121 GTCCCCGCTGCAGATCTCT 122 CTCCAGCTTAGGGTAGTTGTCCAT 123 CGGATCCTTTCCTCACTCGCCCA EGR3 NM_004430.2 125 CCATGTGGATGAATGAGGTG 126 TGCCTGAGAAGAGGTGAGGT 127 ACCCAGTCTCACCTTCTCCCCACC EGR NM_004449.3 129 CCAACACTAGGCTCCCCA 130 CCTCCGCCAGGTCTTTAGT 131 AGCCATATGCCTTCTCATCTGGGC F2R NM_001992.2 133 AAGGAGCAAACCATCCAGG 134 GCAGGGTTTCATTGAGCAC 135 CCCGGGCTCAACATCACTACCTGT FAM107A NM_007177.2 137 TTCTGCCCAGGCCTTCCCAC 138 AGGAGCTGGGGTGTACGGAGA 139 TCTCCGAGGCTCCCCAGGGCCCCG FAM13C NM_198215.2 141 ATCTTCAAAGCGGAGAGCG 142 GCTGGATACCACATGCTCTG 143 TCCTGACTTTCTCCGTGGCTCCTC FAP NM_004460.2 145 GTTGGCTCACGTGGGTTAC 146 GACAGGACCGAAACATTCTG 147 AGCCACTGCAAACATACTCGTTCATCA FLNC NM_001458.4 149 CAGGACAATGGTGATGGCT 150 TGATGGTGTACTCGCCAGG 151 ATGTGCTGTCAGCTACCTGCCCAC FN1 NM_002026.2 153 GGAAGTGACAGACGTGAAGGT 154 ACACGGTAGCCGGTCACT 155 ACTCTCAGGCGGTGTCCACATGAT FOS NM_005252.2 157 CGAGCCCTTTGATGACTTCCT 158 GGAGCGGGCTGTCTCAGA 159 TCCCAGCATCATCCAGGCCCAG GADD45B NM_015675.1 161 ACCCTCGACAAGACCACACT 162 TGGGAGTTCATGGGTACAGA 163 TGGGAGTTCATGGGTACAGA GPM6B NM_001001994.1 165 ATGTGCTTGGAGTGGCCT 166 TGTAGAACATAAACACGGGCA 167 CGCTGAGAAACCAAACACACCCAG GPS1 NM_004127.4 169 AGTACAAGCAGGCTGCCAAG 170 GCAGCTCAGGGAAGTCACA 171 CCTCCTGCTGGCTTCCTTTGATCA GSN NM_000177.1 173 CTTCTGCTAAGCGGTACATCGA 174 GGCTCAAAGCCTTGCTTCAC 175 ACCCAGCCAATCGGGATCGGC GSTM1 NM_000561.1 177 AAGCTATGAGGAAAAGAAGTACACGAT 178 GGCCCAGCTTGAATTTTTCA 179 TCAGCCACTGGCTTCTGTCATAATCAGGAG GSTM2 NM_000848.2 181 CTGCAGGCACTCCCTGAAAT 182 CCAAGAAACCATGGCTGCTT 183 CTGAAGCTCTACTCACAGTTTCTGGG HLF NM_002126.4 185 CACCCTGCAGGTGTCTGAG 186 GGTACCTAGGAGCAGAAGGTGA 187 TAAGTGATCTGCCCTCCAGGTGGC IGF1 NM_000618.1 189 TCCGGAGCTGTGATCTAAGGA 190 CGGACAGAGCGAGCTGACTT 191 TGTATTGCGCACCCCTCAAGCCTG IGFBP2 NM_000597.1 193 GTGGACAGCACCATGAACA 194 CCTTCATACCCGACTTGAGG 195 CTTCCGGCCAGCACTGCCTC IGFBP6 NM_002178.1 197 TGAACCGCAGAGACCAACAG 198 GTCTTGGACACCCGCAGAAT 199 ATCCAGGCACCTCTACCACGCCCTC IL6ST NM_002184.2 201 GGCCTAATGTTCCAGATCCT 202 AAAATTGTGCCTTGGAGGAG 203 CATATTGCCCAGTGGTCACCTCACA INHBA NM_002192.1 205 GTGCCCGAGCCATATAGCA 206 CGGTAGTGGTTGATGACTGTTGA 207 ACGTCCGGGTCCTCACTGTCCTTCC ITGA7 NM_002206.1 209 GATATGATTGGTCGCTGCTTTG 210 AGAACTTCCATTCCCCACCAT 211 CAGCCAGGACCTGGCCATCCG JUN NM_002228.2 213 GACTGCAAAGATGGAAACGA 214 TAGCCATAAGGTCCGCTCTC 215 CTATGACGATGCCCTCAACGCCTC KLK2 NM_005551.3 217 AGTCTCGGATTGTGGGAGG 218 TGTACACAGCCACCTGCC 219 TTGGGAATGCTTCTCACACTCCCA KRT15 NM_002275.2 221 GCCTGGTTCTTCAGCAAGAC 222 CTTGCTGGTCTGGATCATTTC 223 TGAACAAAGAGGTGGCCTCCAACA KRT5 NM_000424.2 225 TCAGTGGAGAAGGAGTTGGA 226 TGCCATATCCAGAGGAAACA 227 CCAGTCAACATCTCTGTTGTCACAAGCA LAMB3 NM_000228.1 229 ACTGACCAAGCCTGAGACCT 230 GTCACACTTGCAGCATTTCA 231 CCACTCGCCATACTGGGTGCAGT LGALS3 NM_002306.1 233 AGCGGAAAATGGCAGACAAT 234 CTTGAGGGTTTGGGTTTCCA 235 ACCCAGATAACGCATCATGGAGCGA MMP11 NM_005940.2 237 CCTGGAGGCTGCAACATACC 238 TACAATGGCTTTGGAGGATAGCA 239 ATCCTCCTGAAGCCCTTTTCGCAGC MYBL2 NM_002466.1 241 GCCGAGATCGCCAAGATG 242 CTTTTGATGGTAGAGTTCCAGTGATTC 243 CAGCATTGTCTGTCCTCCCTGGCA NFAT5 NM_006599.2 245 CTGAACCCCTCTCCTGGTC 246 AGGAAACGATGGCGAGGT 247 CGAGAATCAGTCCCCGTGGAGTTC OLFML3 NM_020190.2 249 TCAGAACTGAGGCCGACAC 250 CCAGATAGTCTACCTCCCGCT 251 CAGACGATCCACTCTCCCGGAGAT PAGE4 NM_007003.2 253 GAATCTCAGCAAGAGGAACCA 254 GTTCTTCGATCGGAGGTGTT 255 CCAACTGACAATCAGGATATTGAACCTGG PGK1 NM_000291.1 257 AGAGCCAGTTGCTGTAGAACTCAA 258 CTGGGCCTACACAGTCCTTCA 259 TCTCTGCTGGGCAAGGATGTTCTGTTC PPAP2B NM_003713.3 261 ACAAGCACCATCCCAGTGA 262 CACGAAGAAAACTATGCAGCAG 263 ACCAGGGCTCCTTGAGCAAATCCT PPP1R12A NM_002480.1 265 CGGCAAGGGGTTGATATAGA 266 TGCCTGGCATCTCTAAGCA 267 CCGTTCTTCTTCCTTTCGAGCTGC PRKCA NM_002737.1 269 CAAGCAATGCGTCATCAATGT 270 GTAAATCCGCCCCCTCTTCT 271 CAGCCTCTGCGGAATGGATCACACT SDC1 NM_002997.1 273 GAAATTGACGAGGGGTGTCT 274 AGGAGCTAACGGAGAACCTG 275 CTCTGAGCGCCTCCATCCAAGG SFRP4 NM_003014.2 277 TACAGGATGAGGCTGGGC 278 GTTGTTAGGGCAAGGGGC 279 CCTGGGACAGCCTATGTAAGGCCA SHMT2 NM_005412.4 281 AGCGGGTGCTAGAGCTTGTA 282 ATGGCACTTCGGTCTCCA 283 CCATCACTGCCAACAAGAACACCTG SLC22A3 NM_021977.2 285 ATCGTCAGCGAGTTTGACCT 286 CAGGATGGCTTGGGTGAG 287 CAGCATCCACGCATTGACACAGAC SMAD4 NM_005359.3 289 GGACATTACTGGCCTGTTCACA 290 ACCAATACTCAGGAGCAGGATGA 291 TGCATTCCAGCCTCCCATTTCCA SPARC NM_003118.1 293 TCTTCCCTGTACACTGGCAGTTC 294 AGCTCGGTGTGGGAGAGGTA 295 TGGACCAGCACCCCATTGACGG SRC NM_005417.3 297 TGAGGAGTGGTATTTTGGCAAGA 298 CTCTCGGGTTCTCTGCATTGA 299 AACCGCTCTGACTCCCGTCTGGTG SRD5A2 NM_000348.2 301 GTAGGTCTCCTGGCGTTCTG 302 TCCCTGGAAGGGTAGGAGTAA 303 AGACACCACTCAGAATCCCCAGGC STAT5B NM_012448.1 305 CCAGTGGTGGTGATCGTTCA 306 GCAAAAGCATTGTCCCAGAGA 307 CAGCCAGGACAACAATGCGACGG TGFB1I1 NM_001042454.1 309 GCTACTTTGAGCGCTTCTCG 310 GGTCACCATCTTGTGTCGG 311 CAAGATGTGGCTTCTGCAACCAGC THBS2 NM_003247.2 313 CAAGACTGGCTACATCAGAGTCTTAGTG 314 CAGCGTAGGTTTGGTCATAGATAGG 315 TGAGTCTGCCATGACCTGTTTTCCTTCAT TNFRSF10B NM_003842.2 317 CTCTGAGACAGTGCTTCGATGACT 318 CCATGAGGCCCAACTTCCT 319 CAGACTTGGTGCCCTTTGACTCC TPM2 NM_213674.1 321 AGGAGATGCAGCTGAAGGAG 322 CCACCTCTTCATATTTGCGG 323 CCAAGCACATCGCTGAGGATTCAG TPX2 NM_012112.2 325 TCAGCTGTGAGCTGCGGATA 326 ACGGTCCTAGGTTTGAGGTTAAGA 327 CAGGTCCCATTGCCGGGCG TUBB2A NM_001069.1 329 CGAGGACGAGGCTTAAAAAC 330 ACCATGCTTGAGGACAACAG 331 TCTCAGATCAATCGTGCATCCTTAGTGAA UBE2T NM_014176.1 333 TGTTCTCAAATTGCCACCAA 334 AGAGGTCAACACAGTTGCGA 335 AGGTGCTTGGAGACCATCCCTCAA VCL NM_003373.2 337 GATACCACAACTCCCATCAAGCT 338 TCCCTGTTAGGCGCATCAG 339 AGTGGCAGCCACGGCGCC ZFP36 NM_003407.1 341 CATTAACCCACTCCCCTGA 342 CCCCCACCATCATGAATACT 343 CAGGTCCCCAAGTGTGCAAGCTC Official Symbol: SEQ ID NO: Amplicon Sequence: ALDH1A2   4 CACGTCTGTCCCTCTCTGCTTTCTCTGTAGGGCCCAGCTCTCAGGAATACAAAGTTGAGCCACGGTC ANPEP   8 CCACCTTGGACCAAAGTAAAGCGTGGAATCGTTACCGCCTCCCCAACACGCTGAAACCCGATTCCTACCGGGTGACGCTGAGA AR  12 CGACTTCACCGCACCTGATGTGTGGTACCCTGGCGGCATGGTGAGCAGAGTGCCCTATCCCAGTCCCACTTGTGTCA ARF1  16 CAGTAGAGATCCCCGCAACTCGCTTGTCCTTGGGTCACCCTGCATTCCATAGCCATGTGCTTGT ASPN  20 CATTGCCACTTCAACTCTAAGGAATATTTTTGAGATATCCCTTTGGAAGACCTTGCTTGGAAGAGCCTGGACACTAACAAT ATP5E  24 CCGCTTTCGCTACAGCATGGTGGCCTACTGGAGACAGGCTGGACTCAGCTACATCCGATACTCCCA AZGP1  28 GAGGCCAGCTAGGAAGCAAGGGTTGGAGGCAATGTGGGATCTCAGACCCAGTAGCTGCCCTTCCTG BGN  32 GAGCTCCGCAAGGATGACTTCAAGGGTCTCCAGCACCTCTACGCCCTCGTCCTGGTGAACAACAAG BIN1  36 CCTGCAAAAGGGAACAAGAGCCCTTCGCCTCCAGATGGCTCCCCTGCCGCCACCCCCGAGATCAGAGTCAACCACG BMP6  40 GTGCAGACCTTGGTTCACCTTATGAACCCCGAGTATGTCCCCAAACCGTGCTGTGCGCCAACTAAG C7  44 ATGTCTGAGTGTGAGGCGGGCGCTCTGAGATGCAGAGGGCAGAGCATCTCTGTCACCAGCATAAGGCCT CADM1  48 CCACCACCATCCTTACCATCATCACAGATTCCCGAGCAGGTGAAGAAGGCTCGATCAGGGCAGTGGATC CD276  52 CCAAAGGATGCGATACACAGACCACTGTGCAGCCTTATTTCTCCAATGGACATGATTCCCAAGTCATCC CD44  56 GGCACCACTGCTTATGAAGGAAACTGGAACCCAGAAGCACACCCTCCCCTCATTCACCATGAGCATC CDC20  60 AGTGACCTGCACTCGCTGCTTCAGCTGGATGCACCCATCCCCAATGCACCCCCTGCGCGCTGGCAGCGCAAAGCCAAGGAAGCC CDKN2C  64 TGAAGGGAACCTGCCCTTGCACTTGGCTGCCAAAGAAGGCCACCTCCGGGTGGTGGAGTTCCTGGTGAAGCACA CLTC  68 ACCGTATGGACAGCCACAGCCTGGCTTTGGGTACAGCATGTGAGATGAAGCGCTGATCCTGTAGTCA COL1A1  72 GTGGCCATCCAGCTGACCTTCCTGCGCCTGATGTCCACCGAGGCCTCCCAGAACATCACCTACCACTG COL1A2  76 CAGCCAAGAACTGGTATAGGAGCTCCAAGGACAAGAAACACGTCTGGCTAGGAGAAACTATCAATGCTGGCAGCCAGTTT COL3A1  80 GGAGGTTCTGGACCTGCTGGTCCTCCTGGTCCCCAAGGTGTCAAAGGTGAACGTGGCAGTCCTGGT COL4A1  84 ACAAAGGCCTCCCAGGATTGGATGGCATCCCTGGTGTCAAAGGAGAAGCAGGTCTTCCTGGGACTC COL5A2  88 GGTCGAGGAACCCAAGGTCCGCCTGGTGCTACAGGATTTCCTGGTTCTGCGGGCAGAGTTGGACCTCCAGGC COL6A1  92 GGAGACCCTGGTGAAGCTGGCCCGCAGGGTGATCAGGGAAGAGAAGGCCCCGTTGGTGTCCCTGGAGA COL8A1  96 TGGTGTTCCAGGGCTTCTCGGACCTAAGGGAGAGCCAGGAATCCCAGGGGATCAGGGTTTACAGGG CSF1 100 TGCAGCGGCTGATTGACAGTCAGATGGAGACCTCGTGCCAAATTACATTTGAGTTTGTAGACCAGGAACAGTTG CSRP1 104 ACCCAAGACCCTGCCTCTTCCACTCCACCCTTCTCCAGGGACCCTTAGATCACATCACTCCACCCCTGC CYP3A5 108 TCATTGCCCAGTATGGAGATGTATTGGTGAGAAACTTGAGGCGGGAAGCAGAGAAAGGCAAGCCTGTC DES 112 ACTTCTCACTGGCCGACGCGGTGAACCAGGAGTTTCTGACCACGCGCACCAACGAGAAGGTGGAGC DPP4 116 GTCCTGGGATCGGGAAGTGGCGTGTTCAAGTGTGGAATAGCCGTGGCGCCTGTATCCCGGTGGGAGTAC DUSP1 120 AGACATCAGCTCCTGGTTCAACGAGGCCATTGACTTCATAGACTCCATCAAGAATGCTGGAGGAAGGGTGTTTGTC EGR1 124 GTCCCCGCTGCAGATCTCTGACCCGTTCGGATCCTTTCCTCACTCGCCCACCATGGACAACTACCCTAAGCTGGAG EGR3 128 CCATGTGGATGAATGAGGTGTCTCCTTTCCATACCCAGTCTCACCTTCTCCCCACCCTACCTCACCTCTTCTCAGGCA ERG 132 CCAACACTAGGCTCCCCACCAGCCATATGCCTTCTCATCTGGGCACTTACTACTAAAGACCTGGCGGAGG F2R 136 AAGGAGCAAACCATCCAGGTGCCCGGGCTCAACATCACTACCTGTCATGATGTGCTCAATGAAACCCTGC FAM107A 140 TTCTGCCCAGGCCTTCCCACCAGGAATCTCCGAGGCTCCCCAGGGCCCCGCTTCTCCGTACACCCCAGCTCCT FAM13C 144 ATCTTCAAAGCGGAGAGCGGGAGGAGCCACGGAGAAAGTCAGGAGACAGAGCATGTGGTATCCAGC FAP 148 GTTGGCTCACGTGGGTTACTGATGAACGAGTATGTTTGCAGTGGCTAAAAAGAGTCCAGAATGTTTCGGTCCTGTC FLNC 152 CAGGACAATGGTGATGGCTCATGTGCTGTCAGCTACCTGCCCACGGAGCCTGGCGAGTACACCATCA FN1 156 GGAAGTGACAGACGTGAAGGTCACCATCATGTGGACACCGCCTGAGAGTGCAGTGACCGGCTACCGTGT FOS 160 CGAGCCCTTTGATGACTTCCTGTTCCCAGCATCATCCAGGCCCAGTGGCTCTGAGACAGCCCGCTCC GADD45B 164 ACCCTCGACAAGACCACACTTTGGGACTTGGGAGCTGGGGCTGAAGTTGCTCTGTACCCATGAACTCCCA GPM6B 168 ATGTGCTTGGAGTGGCCTGGCTGGGTGTGTTTGGTTTCTCAGCGGTGCCCGTGTTTATGTTCTACA GPS1 172 AGTACAAGCAGGCTGCCAAGTGCCTCCTGCTGGCTTCCTTTGATCACTGTGACTTCCCTGAGCTGC GSN 176 CTTCTGCTAAGCGGTACATCGAGACGGACCCAGCCAATCGGGATCGGCGGACGCCCATCACCGTGGTGAAGCAAGGCTTTGAGCC GSTM1 180 AAGCTATGAGGAAAAGAAGTACACGATGGGGGACGCTCCTGATTATGACAGAAGCCAGTGGCTGAATGAAAAATTCAAGCTGGGCC GSTM2 184 CTGCAGGCACTCCCTGAAATGCTGAAGCTCTACTCACAGTTTCTGGGGAAGCAGCCATGGTTTCTTGG HLF 188 CACCCTGCAGGTGTCTGAGACTAAGTGATCTGCCCTCCAGGTGGCGATCACCTTCTGCTCCTAGGTACC IGF1 192 TCCGGAGCTGTGATCTAAGGAGGCTGGAGATGTATTGCGCACCCCTCAAGCCTGCCAAGTCAGCTCGCTCTGTCCG IGFBP2 196 GTGGACAGCACCATGAACATGTTGGGCGGGGGAGGCAGTGCTGGCCGGAAGCCCCTCAAGTCGGGTATGAAGG IGFBP6 200 TGAACCGCAGAGACCAACAGAGGAATCCAGGCACCTCTACCACGCCCTCCCAGCCCAATTCTGCGGGTGTCCAAGAC IL6ST 204 GGCCTAATGTTCCAGATCCTTCAAAGAGTCATATTGCCCAGTGGTCACCTCACACTCCTCCAAGGCACAATTTT INHBA 208 GTGCCCGAGCCATATAGCAGGCACGTCCGGGTCCTCACTGTCCTTCCACTCAACAGTCATCAACCACTACCG ITGA7 212 GATATGATTGGTCGCTGCTTTGTGCTCAGCCAGGACCTGGCCATCCGGGATGAGTTGGATGGTGGGGAATGGAAGTTCT JUN 216 GACTGCAAAGATGGAAACGACCTTCTATGACGATGCCCTCAACGCCTCGTTCCTCCCGTCCGAGAGCGGACCTTATGGCTA KLK2 220 AGTCTCGGATTGTGGGAGGCTGGGAGTGTGAGAAGCATTCCCAACCCTGGCAGGTGGCTGTGTACA KRT15 224 GCCTGGTTCTTCAGCAAGACTGAGGAGCTGAACAAAGAGGTGGCCTCCAACACAGAAATGATCCAGACCAGCAAG KRT5 228 TCAGTGGAGAAGGAGTTGGACCAGTCAACATCTCTGTTGTCACAAGCAGTGTTTCCTCTGGATATGGCA LAMB3 232 ACTGACCAAGCCTGAGACCTACTGCACCCAGTATGGCGAGTGGCAGATGAAATGCTGCAAGTGTGAC LGALS3 236 AGCGGAAAATGGCAGACAATTTTTCGCTCCATGATGCGTTATCTGGGTCTGGAAACCCAAACCCTCAAG MMP11 240 CCTGGAGGCTGCAACATACCTCAATCCTGTCCCAGGCCGGATCCTCCTGAAGCCCTTTTCGCAGCACTGCTATCCTCCAAAGCCATTGTA MYBL2 244 GCCGAGATCGCCAAGATGTTGCCAGGGAGGACAGACAATGCTGTGAAGAATCACTGGAACTCTACCATCAAAAG NFAT5 248 CTGAACCCCTCTCCTGGTCACCGAGAATCAGTCCCCGTGGAGTTCCCCCTCCACCTCGCCATCGTTTCCT OLFML3 252 TCAGAACTGAGGCCGACACCATCTCCGGGAGAGTGGATCGTCTGGAGCGGGAGGTAGACTATCTGG PAGE4 256 GAATCTCAGCAAGAGGAACCACCAACTGACAATCAGGATATTGAACCTGGACAAGAGAGAGAAGGAACACCTCCGATCGAAGAAC PGK1 260 AGAGCCAGTTGCTGTAGAACTCAAATCTCTGCTGGGCAAGGATGTTCTGTTCTTGAAGGACTGTGTAGGCCCAG PPAP2B 264 ACAAGCACCATCCCAGTGATGTTCTGGCAGGATTTGCTCAAGGAGCCCTGGTGGCCTGCTGCATAGTTTTCTTCGTG PPP1R12A 268 CGGCAAGGGGTTGATATAGAAGCAGCTCGAAAGGAAGAAGAACGGATCATGCTTAGAGATGCCAGGCA PRKCA 272 CAAGCAATGCGTCATCAATGTCCCCAGCCTCTGCGGAATGGATCACACTGAGAAGAGGGGGCGGATTTAC SDC1 276 GAAATTGACGAGGGGTGTCTTGGGCAGAGCTGGCTCTGAGCGCCTCCATCCAAGGCCAGGTTCTCCGTTAGCTCCT SFRP4 280 TACAGGATGAGGCTGGGCATTGCCTGGGACAGCCTATGTAAGGCCATGTGCCCCTTGCCCTAACAAC SHMT2 284 AGCGGGTGCTAGAGCTTGTATCCATCACTGCCAACAAGAACACCTGTCCTGGAGACCGAAGTGCCAT SLC22A3 288 ATCGTCAGCGAGTTTGACCTTGTCTGTGTCAATGCGTGGATGCTGGACCTCACCCAAGCCATCCTG SMAD4 292 GGACATTACTGGCCTGTTCACAATGAGCTTGCATTCCAGCCTCCCATTTCCAATCATCCTGCTCCTGAGTATTGGT SPARC 296 TCTTCCCTGTACACTGGCAGTTCGGCCAGCTGGACCAGCACCCCATTGACGGGTACCTCTCCCACACCGAGCT SRC 300 TGAGGAGTGGTATTTTGGCAAGATCACCAGACGGGAGTCAGAGCGGTTACTGCTCAATGCAGAGAACCCGAGAG SRD5A2 304 GTAGGTCTCCTGGCGTTCTGCCAGCTGGCCTGGGGATTCTGAGTGGTGTCTGCTTAGAGTTTACTCCTACCCTTCCAGGGA STAT5B 308 CCAGTGGTGGTGATCGTTCATGGCAGCCAGGACAACAATGCGACGGCCACTGTTCTCTGGGACAATGCTTTTGC TGFB1I1 312 GCTACTTTGAGCGCTTCTCGCCAAGATGTGGCTTCTGCAACCAGCCCATCCGACACAAGATGGTGACC THBS2 316 CAAGACTGGCTACATCAGAGTCTTAGTGCATGAAGGAAAACAGGTCATGGCAGACTCAGGACCTATCTATGACCAAACCTACGCTG TNFRSF10B 320 CTCTGAGACAGTGCTTCGATGACTTTGCAGACTTGGTGCCCTTTGACTCCTGGGAGCCGCTCATGAGGAAGTTGGGCCTCATGG TPM2 324 AGGAGATGCAGCTGAAGGAGGCCAAGCACATCGCTGAGGATTCAGACCGCAAATATGAAGAGGTGG TPX2 328 TCAGCTGTGAGCTGCGGATACCGCCCGGCAATGGGACCTGCTCTTAACCTCAAACCTAGGACCGT TUBB2A 332 CGAGGACGAGGCTTAAAAACTTCTCAGATCAATCGTGCATCCTTAGTGAACTTCTGTTGTCCTCAAGCATGGT UBE2T 336 TGTTCTCAAATTGCCACCAAAAGGTGCTTGGAGACCATCCCTCAACATCGCAACTGTGTTGACCTCT VCL 340 GATACCACAACTCCCATCAAGCTGTTGGCAGTGGCAGCCACGGCGCCTCCTGATGCGCCTAACAGGGA ZFP36 344 CATTAACCCACTCCCCTGACCTCACGCTGGGGCAGGTCCCCAAGTGTGCAAGCTCAGTATTCATGATGGTGGGGG 

What is claimed is:
 1. A method of analyzing a biological sample containing cancer cells from a patient with prostate cancer, comprising: determining a level of RNA transcripts of each of a set of genes consisting of: BGN, COL1A1, SFRP4, FLNC, GSN, GSTM2, TPM2, AZGP1, KLK2, FAM13C, SRD5A2, and TPX2, and one or more reference genes, in the biological sample, normalizing the level of the RNA transcripts against the level of the one or more reference RNA transcripts from the sample to obtain normalized expression levels of the BGN, COL1A1, SFRP4, FLNC, GSN, GSTM2, TPM2, AZGP1, KLK2, FAM13C, SRD5A2, and TPX2 RNA transcripts, assigning the RNA transcripts to gene groups selected from a cellular organization gene group, an androgen gene group, a stromal response gene group, and a proliferation gene group; wherein the cellular organization group comprises FLNC, GSN, TPM2, and GSTM2, the androgen group comprises FAM13C, KLK2, AZGP1 and SRD5A2, the stromal response group comprises BGN, COL1A1, and SFRP4, and the proliferation group comprises TPX2, and calculating one or more quantitative scores selected from (a) a stromal response group score obtained from normalized expression levels of the stromal response group genes wherein the stromal group score equals 0.527X BGN+0.457 X COL1A1+0.156 X SFRP4, (b) a cellular organization group score obtained from normalized expression levels of the cellular organization group genes wherein the cellular organization group score equals 0.163 X FLNC+0.504 X GSN+0.421 X TPM2+0.394 X GSTM2, (c) an androgen group score obtained from normalized expression levels of the androgen group genes wherein the androgen group score equals 0.634 X FAM13C+1.079 X KLK2+0.642 X AZGP1+0.997 X SRD5A2 threshold score where the SRD5A2 threshold score equals SRD5A2 if SRD5A2 is 5.5 or higher and equals 5.5 if SRD5A2 is less than 5.5, and (d) a TPX2 threshold score wherein the TPX2 threshold score equals TPX2 if TPX2 is 5.0 or higher and equals 5.0 if TPX2 is less than 5.0, wherein the gene symbols represent the normalized gene expression levels of the RNA transcripts of the respective genes.
 2. The method of claim 1, wherein the biological sample is a tissue sample.
 3. The method of claim 2, wherein the tissue sample is fixed, paraffin-embedded, or fresh, or frozen.
 4. The method of claim 1, wherein the level of one or more RNA transcripts is determined by quantitative RT-PCR.
 5. The method of claim 1, further comprising creating a report providing the one or more quantitative score results.
 6. The method of claim 1, wherein the one or more reference transcripts comprise RNA transcripts from one or more of ARF1, ATP5E, CLTC, GPS1, and PGK1.
 7. A method of treating a prostate cancer patient, comprising: administering active surveillance to the patient; wherein the patient was previously determined to have a low or intermediate risk of adverse pathology, non-organ-confined disease, high-grade disease, or high-grade or non-organ confined disease according to an analysis comprising: determining a level of RNA transcripts of each of a set of genes consisting of: BGN, COL1A1, SFRP4, FLNC, GSN, GSTM2, TPM2, AZGP1, KLK2, FAM13C, SRD5A2, and TPX2, and one or more reference genes, in a biological sample from the patient, normalizing the level of the RNA transcripts against the level of the one or more reference RNA transcripts from the sample to obtain normalized expression levels of the BGN, COL1A1, SFRP4, FLNC, GSN, GSTM2, TPM2, AZGP1, KLK2, FAM13C, SRD5A2, and TPX2 RNA transcripts, comparing the normalized expression levels to gene expression data in reference prostate cancer samples, wherein increased normalized expression levels of BGN, COL1A1, SFRP4, and TPX2 correlate with an increased likelihood of adverse pathology, non-organ-confined disease, high-grade disease, or high-grade or non-organ confined disease, and wherein increased normalized expression levels of FLNC, GSN, GSTM2, TPM2, AZGP1, KLK2, FAM13C, and SRD5A2 correlate with a decreased likelihood of adverse pathology, non-organ-confined disease, high-grade disease, or high-grade or non-organ confined disease, assigning the RNA transcripts to gene groups selected from a cellular organization gene group, an androgen gene group, a stromal response gene group, and a proliferation gene group; wherein the cellular organization group comprises FLNC, GSN, TPM2, and GSTM2, the androgen group comprises FAM13C, KLK2, AZGP1 and SRD5A2, the stromal response group comprises BGN, COL1A1, and SFRP4, and the proliferation group comprises TPX2, calculating one or more quantitative scores for the patient, and predicting the likelihood of one or more of adverse pathology, non-organ-confined disease, high-grade disease, or high-grade or non-organ-confined disease for the patient based on the one or more quantitative scores; wherein the one or more quantitative scores are selected from a stromal response group score obtained from normalized expression levels of the stromal response group genes wherein the stromal group score equals 0.527 X BGN+0.457 X COL1A1+0.156 X SFRP4, a cellular organization group score obtained from normalized expression levels of the cellular organization group genes wherein the cellular organization group score equals 0.163 X FLNC+0.504 X GSN+0.421 X TPM2+0.394 X GSTM2, an androgen group score obtained from normalized expression levels of the androgen group genes wherein the androgen group score equals 0.634 X FAM13C+1.079 X KLK2+0.642 X AZGP1+0.997 X SRD5A2 threshold score where the SRD5A2 threshold score equals SRD5A2 if SRD5A2 is 5.5 or higher and equals 5.5 if SRD5A2 is less than 5.5, and a TPX2 threshold score wherein the TPX2 threshold score equals TPX2 if TPX2 is 5.0 or higher and equals 5.0 if TPX2 is less than 5.0, wherein the gene symbols represent the normalized gene expression levels of the RNA transcripts of the respective genes; and wherein the stromal response group score and the proliferation group score positively correlate with an likelihood of adverse pathology, non-organ-confined disease, high-grade disease, and high-grade or non-organ confined disease, and wherein cellular organization group score and androgen group score negatively correlate with a likelihood of adverse pathology, non-organ-confined disease, high-grade disease, and high-grade or non-organ confined disease.
 8. The method of claim 1, wherein the method comprises determining the androgen group score.
 9. The method of claim 7, wherein the method comprises determining the androgen group score.
 10. The method of claim 1, wherein the method comprises determining the stromal group score.
 11. The method of claim 7, wherein the method comprises determining the stromal group score.
 12. The method of claim 1, wherein the method comprises determining the proliferation group score.
 13. The method of claim 7, wherein the method comprises determining the proliferation group score. 